A study of intracellular orthophosphate concentration in human muscle and erythrocytes by 31P nuclear magnetic resonance spectroscopy and selective chemical assay

In order to study the relationship between extracellular and intracellular concentrations of orthophosphate (Pi), phosphorus nuclear magnetic resonance spectra were recorded, at rest, from the flexor digitorum superficialis muscle of hypophosphataemic patients with vitamin D-resistant rickets, and patients with Paget's disease of bone before and after they had been made hyperphosphataemic by treatment with the drug ethylidene-1-hydroxy-1,1-bisphosphonate. Changes in intramuscular P1 were estimated from the ratio of the areas of the Pi to adenosine 5'-triphosphate peaks. Even though the plasma Pi concentration in these patients spanned a fourfold range (0.5-2.0 mmol/l) the corresponding intramuscular Pi concentration increased by only 70%. A similar effect was observed in erythrocytes, from patients with these disorders, which were incubated in autologous plasma at 37 degrees C, under an atmosphere of O2 + CO2 (95:5, v/v). However, chloride ions, which are transported passively across the cell membrane, showed no change in distribution between cells and plasma, indicating that there was no general effect on passive anion distribution. When erythrocytes from normal subjects were incubated in autologous plasma (1.0 mmol of Pi/l) and in plasma supplemented with Pi (2.3 mmol of Pi/l), the Pi concentration in the cells, at steady state, increased only from 0.57 to 0.78 mmol/l cells, suggesting that the effect was not an artifact of disease or drug therapy. It is concluded that, in human skeletal myocytes and erythrocytes, the percentage change in the concentration of cytoplasmic Pi is lower than that in plasma. This implies that these cells can buffer or regulate cytoplasmic Pi when the extracellular concentration is disturbed.     

Necrotizing fasciitis in a renal transplant patient

We have described a 28-year-old diabetic woman who had necrotizing fasciitis of the perineum three years after receiving a living related renal transplant. The diagnosis of necrotizing fasciitis was made early and she was referred to a tertiary care center where she received radical perineal debridement and aggressive medical and surgical follow-up. Necrotizing fasciitis in a transplant patient is rare; review of the literature shows few cases and no survivors. Our patient has returned to a normal life despite continuation of all immunosuppressive therapy throughout the entire hospital course. In addition, she had a good cosmetic result despite the large necrotic perineal infection. Her survival can be attributed to early diagnosis and referral, immediate and extensive debridement, and aggressive protein replacement.     

Renal preservation technics

This paper discusses modern renal preservation technics, presenting historic background, advantages, and clinical methods. The monitoring of perfused kidneys, methods of in situ hypothermia, the uses of renal preservation in extracorporeal surgery, and the results of cadaver kidney preservation at the Medical University of South Carolina are highlighted.     

Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability.

Vulnerable areas of atherosclerotic plaques often contain lipid-laden macrophages and display matrix metalloproteinase activity. We hypothesized that reactive oxygen species released by macrophage-derived foam cells could trigger activation of latent proforms of metalloproteinases in the vascular interstitium. We showed that in vivo generated macrophage foam cells produce superoxide, nitric oxide, and hydrogen peroxide after isolation from hypercholesterolemic rabbits. Effects of these reactive oxygens and that of peroxynitrite, likely to result from simultaneous production of nitric oxide and superoxide, were tested in vitro using metalloproteinases secreted by cultured human vascular smooth muscle cells. Enzymes in culture media or affinity-purified (pro-MMP-2 and MMP-9) were examined by SDS-PAGE zymography, Western blotting, and enzymatic assays. Under the conditions used, incubation with xanthine/xanthine oxidase increased the amount of active gelatinases, while nitric oxide donors had no noticeable effect. Incubation with peroxynitrite resulted in nitration of MMP-2 and endowed it with collagenolytic activity. Hydrogen peroxide treatment showed a catalase-reversible biphasic effect (gelatinase activation at concentrations of 4 microM, inhibition at > or = 10-50 microM). Thus, reactive oxygen species can modulate matrix degradation in areas of high oxidant stress and could therefore contribute to instability of atherosclerotic plaques.     

Differential toxicity profile of ricin isoforms correlates with their glycosylation levels

Ricin is one of the most potent and deadly plant toxins from the seeds of Ricinus communis. In view of its high toxicity, ricin is being used as an immunotoxin in cancer therapy. Ricin also has several isoforms with differential glycosylation depending on the seed variety. Our study shows three isoforms designated 1, 2 and 3, which differed in their surface charge, resulting in a different behavior on cation exchange chromatography, two dimensional (pI 5.5-8.7) and native PAGE. The molecular masses of isoform-1, 2 and 3 were measured as 63.55 kDa, 64.03 kDa and 62.8 kDa, respectively, by MALDI-TOF/MS. In vitro studies with monkey kidney (Vero) cells showed a time dependent increase in cytotoxicity of the isoforms evaluated by extracellular lactate dehydrogenase activity and mitochondrial dehydrogenase assay. These isoforms also induce oxidative stress and DNA damage. Among the isoforms, isoform-3 was quick to generate reactive oxygen species (ROS), (in 90 min) and exhibited maximum cytotoxicity. Morphological changes, catalase activity and DNA fragmentation were significantly higher with isoform-3 treatment compared to others. The glycosylation studies by MALDI-TOF/MS showed that isoform-3 is highly glycosylated with high sugar levels containing more of hybrid/complex type glycopeptides with mannose as hexose units. These experimental evidences clearly suggest that isoform-3 is superior in its early ROS generation, potency to induce oxidative stress and cytotoxicity, that could be due to it's higher glycosylation levels which make isoform-3 as an ideal candidate for immunotoxin studies.     

HLA-DR3 restricted T cell epitope mimicry in induction of autoimmune response to lupus-associated antigen SmD

Although systemic lupus erythematosus (SLE) is a multigenic autoimmune disorder, HLA-D is the most dominant genetic susceptibility locus. This study was undertaken to investigate the hypothesis that microbial peptides bind HLA-DR3 and activate T cells reactive with lupus autoantigens. Using HLA-DR3 transgenic mice and lupus-associated autoantigen SmD protein, SmD_79-93 was identified to contain a dominant HLA-DR3 restricted T cell epitope. This T cell epitope was characterized by using a T-T hybridoma, C1P2, generated from SmD immunized HLA-DR3 transgenic mouse. By pattern search analysis, 20 putative mimicry peptides (P2-P21) of SmD_79-93, from microbial and human origin were identified. C1P2 cells responded to SmD, SmD_79-93 and a peptide (P20) from Vibro cholerae. Immunization of HLA-DR3 mice with P20 induced T cell responses and IgG antibodies to SmD that were not cross-reactive with the immunogen. A T-T hybridoma, P20P1, generated from P20 immunized mice, not only responded to P20 and SmD_79-93, but also to peptides from Streptococcus agalactiae (P17) and human-La related protein (P11). These three T cell mimics (P20, P11 and P17) induced diverse and different autoantibody response profiles. Our data demonstrates for the first time molecular mimicry at T cell epitope level between lupus-associated autoantigen SmD and microbial peptides. Considering that distinct autoreactive T cell clones were activated by different microbial peptides, molecular mimicry at T cell epitope level can be an important pathway for the activation of autoreactive T cells resulting in the production of autoantibodies. In addition, the novel findings reported herein may have significant implications in the pathogenesis of SLE.     

Blood pressure and vascular effects of leptin in humans

Background: Leptin may play a role in mediating obesity-related hypertension. However, its effects on the vasculature and blood pressure (BP) remain poorly defined in humans. Methods: In the first study, we performed a short-term, placebo-controlled, randomized, double-blind, cross-over experiment investigating the actions of recombinant human leptin (r-metHuLeptin) in 15 nonobese adults. To compliment the acute study, we retrospectively analyzed available BP results from a previously performed 85-day, placebo-controlled, randomized, double-blind, parallel weight-loss study using r-metHuLeptin in 284 obese adults. Results: In the acute study, conduit artery endothelial function determined by brachial flow-mediated dilatation (FMD) increased 2 hours following 0.2 mg . Kg(1) subcutaneously (SC) of r-metHuLeptin versus placebo (+3.3% versus -2.8%, P = .02). BP remained unchanged 4 hours after injections. In the retrospective analysis of the weight loss study data, 10 mg every day before noon (QAM), 10 mg every day after noon (QPM), or 10 mg twice a day (BID) SC of r-metHuLeptin was found to not alter the degree of weight loss (-3.2 +/- 3.7 versus -2.9 +/- 3.2 Kg, P = .54), change in systolic (-1.6 + 12.9 versus -2.0 +/- 13.9 mmHg, P = .85) and diastolic BP (-0.2 +/- 8.7 versus -1.5 +/- 8.6, P = .30), as well as heart rate (-1.4 +/- 10.7 versus -1.4 +/- 10.4 beats/min, P = .98) compared to placebo. Conclusions: In our acute study, marked hyperleptinemia rapidly enhanced endothelial function and did not alter BP. The available data from a longer-term study in healthy obese adults did not demonstrate a significant effect of hyperleptinemia upon BP. These combined findings do not support a direct role for leptin in linking obesity to hypertension, however more studies are required to corroborate these observations.     

Medication use, service utilization, and medical costs associated with new episodes of bipolar disorder: evidence from a retrospective claims database

Objective: To identify resource use patterns and costs incurred during new episodes of bipolar disorder.Method: Researchers examined Medstat MarketScan databases covering the interval of January 1, 1998, to December 31, 2002, to identify 6148 patients with new episodes of bipolar disorder as defined by the International Classification of Diseases, Ninth Revision. Resource utilization patterns and costs for the 6 months prior to the index date and for the 30 days and 1 year after the index date were examined. Differences among subcategories of bipolar patients in terms of the resources used before and after the index date were also examined.Results: The majority of the individuals in the study were female (61.3%) and the mean age was 41.9 years. Patients diagnosed as manic had higher bipolar treatment costs (p < .01) and were more likely to be hospitalized for mental health diagnoses 30 days (p < .01) and 1 year after the index diagnosis (p = .02) compared with individuals diagnosed as depressive, mixed, or other/ unknown. Median total medical costs in the 1 year after the index date were highest for those diagnosed as depressed (p = .02), while patients diagnosed as mixed bipolar had significantly more psychiatrist visits after the index date (p < .01). Approximately 15% of patients were not treated with any central nervous system medication, and over 50% of patients were treated with antidepressants.Conclusion: The subcategory of bipolar disorder that an individual is diagnosed as having significantly affects resource use and costs after such diagnosis. Patients diagnosed as manic generally used more resources than other individuals. In addition, results suggest that a large proportion of individuals are not being treated in accordance with recommended treatment guidelines.     

Pulmonary arterial hypertension: noninvasive detection with phase-contrast MR imaging

PURPOSE: To retrospectively identify pulmonary arterial (PA) flow parameters measured with phase-contrast magnetic resonance (MR) imaging that allow noninvasive diagnosis of chronic PA hypertension (PAH). MATERIALS AND METHODS: The study was HIPAA compliant and was approved by the institutional review board; a waiver of informed consent was obtained. Fifty-nine patients (49 female patients; mean age, 46 years; range, 16-85 years) known to have or suspected of having PAH underwent breath-hold phase-contrast MR imaging and right-sided heart catheterization (RHC). The presence of PAH (mean pulmonary artery pressure [mPAP], >25 mm Hg) was confirmed in 42 patients. Parameters, including PA areas, PA strain, average velocity, peak velocity, acceleration time, and ejection time, were measured in each patient by investigators blinded to RHC results. These measurements were correlated with mPAP, systolic pulmonary artery pressure (sPAP), and pulmonary vascular resistance index (PVRI). The diagnostic ability of phase-contrast MR imaging to depict PAH was quantified. Statistical tests included Spearman rho coefficients, receiver operating characteristic curve analysis, and Bland-Altman plots. RESULTS: Results showed average velocity to have the best correlation with mPAP, sPAP, and PVRI (r = -0.73, -0.76, and -0.86, respectively; P < .001). Average velocity (cutoff value = 11.7 cm/sec) revealed PAH with a sensitivity of 92.9% (39 of 42) and a specificity of 82.4% (14 of 17). Sensitivity and specificity for the minimum PA area (cutoff value = 6.6 cm(2)) were 92.9% (39 of 42) and 88.2% (15 of 17), respectively. CONCLUSION: The average blood velocity throughout the cardiac cycle is strongly correlated with pulmonary pressures and resistance.