Mitomycin-induced hemolytic uremic syndrome (HUS) is a life-threatening complication of this therapy, and increased levels of circulating immune complexes and hypocomplementemia have been found in some patients. We further characterize the role of immune complexes in mitomycin-HUS by showing that removal of these complexes by immunoadsorption with staphylococcal protein A columns correlates with temporal improvement in the microangiopathic hemolytic anemia in this disorder. Two immune complexes bound to the protein A column were identified: an 11S platelet-aggregating protein and a 15S non-platelet-aggregating protein. In addition, the patient had anaphylactoid reactions at the onset of immunoadsorption similar to first-use dialysis reactions that correlated with increases in complement 3a and 5a, interleukin-1 (IL-1), IL-6, and tumor necrosis factor levels. This case suggests that platelet-aggregating and complement-fixing circulating immune complexes are, in part, a proximate cause for mitomycin C-induced HUS. Therapy for mitomycin (HUS) with protein A columns should continue until circulating immune complexes reach undetectable levels and serum complement levels return to the normal range.

Neuropeptide regulation of immunological activity is becoming an important issue in both basic and clinical sciences, necessitating the need for analysis to be performed at the single-cell level. A microsampling procedure has been developed for studying secretion of biologically important peptides from neuropeptide-stimulated lymphocytes, based on microdialysis sampling coupled to immunoaffinity capillary electrophoresis (ICE), with laser-induced fluorescence (LIF) detection using a fibre-optic spectrometer and diode laser excitation. The system demonstrated a limit of detection in the high attomole (10(-18) mol/L) range with pure standards and was capable of monitoring secretion from a single cell over time. Using this system it was possible to differentiate the effects of four neuropeptides on both T and B cell release of regulatory cytokines. CD4(+) lymphocytes demonstrated a 7.5-fold increase in cytokine secretion over baseline following stimulation with substance P (SP) and calcitonin gene-related peptide (CGRP). B cells responded to CGRP and vasoactive intestinal peptide (VIP) stimulation (5.5-fold increase), but not to SP. These changes took place 12--20 h post-stimulation and, once the peak secretion had been reached, remained at that level for the duration of the experiment. This system demonstrates the ability to perform high sensitivity measurements on microsamples of biological fluids.

Objective: To explore the association of neonatal interferons (IFNs) with spastic cerebral palsy (CP) and with other measured substances.Study design: Assays of archived neonatal blood of 31 predominantly term children with CP and 65 children in a control group were obtained by recycling immunoaffinity chromatography with laser-enhanced fluorescence and chemiluminescence detection.Results: Fourteen of 31 children with spastic CP had concentrations of IFNs-alpha, beta, and gamma exceeding any control. Levels of interleukins-1 6, 8, tumor necrosis factor-alpha, chemokines, colony stimulating factors, transforming growth factor-beta, complement components and regulators, certain neuropeptides, and thyroid hormones also differed from control levels in these 14 children. The 17 children with CP whose IFN concentrations were within the control range had levels of inflammatory cytokines higher than but near to control values; 13 of these 17 had values for coagulation factors that exceeded control values. Seven of 9 children with spastic diplegia had high IFNs, and 8 of 10 hemiplegic children had normal IFNs.Conclusion: Neonatal IFNs exceeding control concentrations were associated with other biochemical and clinical indicators of inflammation and with spastic diplegia. In these children with CP, IFNs within the control range were associated with concentrations of other inflammatory markers that were near to control values and with spastic hemiplegia.

Elevated plasma homocysteine levels have been associated with increased atherosclerotic disease risk. Estrogen and estrogen/progestin replacement therapy have been suggested to lower plasma homocysteine levels in postmenopausal women. To assess the impact of hormone replacement therapy (HRT) on plasma homocysteine, levels were measured in samples from adherent women randomized to placebo (n = 34), conjugated equine estrogens (n = 36), or continuous conjugated equine estrogens + progestin (n = 33) in the Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial. Homocysteine levels decreased between baseline and follow-up (12 and 36 months) in all treatment groups. The magnitude of the reduction was greater in the conjugated estrogens group at 12 months compared with placebo (p = 0.036), even when adjusted for folate and B vitamin consumption, but this difference did not persist at 36 months. These data suggest that estrogen therapy has a modest, but transient, impact on plasma homocysteine levels in women with normal homocysteine at baseline.

A micro-sampling procedure has been developed for studying lymphocyte secretion of biologically important peptides in low cell density cultures. The technique is based on microdialysis recovery of the analytes of interest coupled with immunoaffinity capillary electrophoresis separation of the microdialysis samples and laser-induced fluorescence detection. Although the technique is able to recover secreted materials only at the 5–10 cell level, the detection system has a limit of detection (LOD) in the attomole (10−18 M) range. This degree of sensitivity indicates that the system has the potential to measure secreted products at the single cell level. An added advantage of this system over other sampling techniques is that the microdialysis probe allows continuous sampling over time.

Dietary Mg-deficiency in rodents induces a pro-oxidant/proinflammatory condition in vivo characterized by enhanced free radical (lipid radicals and nitric oxide) production, accumulation of oxidation products and pro-oxidant metals, depletion of endogenous antioxidants, and elevated circulating levels of inflammatory mediators. Untreated, this condition leads to cardiomyopathy, as well as reduced myocardial tolerance to imposed oxidative stresses (lipid hydroperoxide exposure; ischemia/reperfusion). The early detection of elevated circulating substance P levels Coy dietary day 3) in this model led to the hypothesis that this neuropeptide may trigger subsequent inflammatory and pro-oxidant events in vivo. Treatment in viva with substance P receptor antagonists (or with select antioxidants) provided substantial protection against pro-oxidant stress, evidenced by the attenuation of blood glutathione loss and cardiac lesion formation in vivo, and postischemic injury in vitro. The protection provided by the receptor antagonists demonstrates a direct contribution of early substance P-mediated inflammation in the subsequent development of the Mg-deficient cardiomyopathy and reduced tolerance to postischemic oxidative stress.

The first week of dietary magnesium deficiency in rodent models is characterized by the induction of raised levels of neuropeptides (substance P [SP] and calcitonin gene related peptide [CGRP]), followed shortly thereafter by inflammatory cytokine release. Since neuropeptides participate in neurogenic inflammation, we have proposed that the neurogenic inflammatory response plays a role in the pathology of magnesium deficiency. However, the association between the early neuropeptide release and the subsequent pathology in this model remains unclear. Peripheral blood T lymphocytes were obtained from Balb/c mice fed a magnesium-deficient diet (approximately 1.8 mmol Mg/kg), or the same diet supplemented with 20 mmol MgO/kg. These cells were incubated in medium containing 10(-10) to 10(-5) M SP, after which the cells were examined for expression of SP receptors and the supernatants were collected and examined by immunochemical techniques for the presence of T lymphocyte associated cytokines. SP stimulation induced the secretion of interleukin (IL)-2, 4, 5, 10, 12, 13 and interferon-gamma (IFN-gamma). T lymphocytes from magnesium-deficient animals, when compared to magnesium-sufficient ones, secreted increased levels of these cytokines. The secretion of these cytokines was maximal at either 5 days (IL-4, IL-5) or 7 days (II-2, IL-10, and IFN-gamma) of magnesium deficiency. This increased sensitivity to SP appears to be related to an increased expression of SP receptors on the surface of T lymphocytes during the first week of magnesium deficiency. These data indicate that SP released early during magnesium deficiency exerts a regulatory role on T lymphocyte cytokine production, especially those cytokines regulating mast cell and immune responses leading to the onset of an immunopathological state.

Neuropeptide modulation of host defenses is associated with a number of clinical conditions in which tissue injury is caused by a process termed neurogenic inflammation. Monitoring of neuropeptide activity during both disease episodes and therapy has proven difficult due to a lack of correlation between laboratory evaluations and disease activity. Although a number of assays are available for measuring the total neuropeptide content of tissue samples, few can measure the bioactive component which is the more physiologically accurate parameter. In an attempt to overcome this problem, a receptor-affinity chromatographic technique coupled with immunological detection has been developed for measuring three bioactive neuropeptides (substance P, vasoactive intestinal peptide, and calcitonin gene-related peptide) in tissue biopsy extracts. The technique involves isolation of the active neuropeptides via their ability to bind to immobilized receptors followed by measurement of the bound materials by a series of simultaneous immunoassays. The techniques compares favourably with results obtained using conventional bioassays and has the added advantage that multiple analytes can be measured in a single procedure.

In previous work we reported the elevation of circulating inflammatory cytokines in rodents maintained on a Mg(2+)-deficient diet. Within the first week of Mg2+ deficiency, significant elevation of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) occurs. The present study was designed to assess the effects of SP receptor blockade by CP-96,945 and its inactive enantiomer CP-96,344 on tissue cytokine levels and in vivo oxidative indexes. CP-96,345 had no significant effect on circulating levels of SP or CGRP; however, at the tissue level, a significant decrease (P < .01) in myocardial accumulation of SP occurred; the inactive enantiomer was only slightly effective. In addition, CP-96,345 significantly reduced (by 53%) the accumulation of tumor necrosis factor-alpha (TNF-alpha) (but not interleukin-1 and interleukin-6) within the lesions; the effect of the enantiomer was insignificant. We conclude that treatment with CP-96,345 inhibits SP and TNF-alpha tissue levels in cardiac lesions, indicating a linkage between this neuropeptide and TNF-alpha. Both SP and TNF-alpha can trigger free radical production; plasma thiobarbituric acid-reactive materials were elevated 2.5-fold and red blood cell reduced glutathione was reduced 55% during Mg2+ deficiency. In the presence of CP-96,345, both indexes of in vivo oxidation were significantly attenuated; the enantiomer was ineffective. These latter observations point to a neuropeptide/TNF-alpha/free radical-triggered mechanism that may be the major pathway of systemic oxidative injury inducing the cardiomyopathic lesions seen during Mg2+ deficiency.