Frequency analysis of IgE-secreting B lymphocytes in persons with normal or elevated serum IgE levels

The immunoregulatory mechanisms that determine the high serum IgE antibody levels in disorders such as helminth parasite infections and the hyper-IgE recurrent infection syndrome (HIE) remain poorly understood. To assess whether elevated serum IgE levels result from an increased number of B lymphocytes committed to IgE production, the proportion of IgE-producing B lymphocytes was determined by a filter immunoplaque assay using PBMC from persons with a broad range of serum IgE levels that included normal persons (n = 9) and patients with loiasis (n = 12), tropical pulmonary eosinophilia (TPE) (n = 6), lymphatic filariasis (n = 28), and HIE (n = 8). PBMC from these persons were assessed for production of in vitro IgE. The geometric mean number of IgE-secreting cells in 10(5) B lymphocytes in PBMC was 0.42 (range 0-2.2) in normal persons, 5.6 (range 0.1-35.5) among patients with loiasis, 9.4 (range 0-53.2) among patients with lymphatic filariasis, 52 (range 31.5-115) among patients with TPE, and 218 (range 56-1404) among patients with HIE. When all study subjects were grouped, there were significant correlations with serum IgE levels (r2 = 0.78; p less than 0.0001) and net spontaneous in vitro IgE production (r2 = 0.8; p less than 0.0001). Estimates of the amount of IgE production per B lymphocyte were similar among normal persons, patients with filarial infections, and patients with TPE (geometric means of 134, 96, and 141 pg/ml/cell, respectively); in contrast, for HIE patients, IgE production by individual B cells was significantly lower (geometric mean 28 pg/ml/cell; p less than 0.001). These observations demonstrate that clonal expansion of IgE-producing B lymphocytes is a major mechanism underlying the elevated serum IgE levels seen in persons with hyper-IgE states.     

Cerium levels are elevated in the serum of patients with endomyocardial fibrosis (EMF)

The geochemical hypothesis on endomyocardial fibrosis (EMF) links causation of the disease to increased levels of cerium in the heart. Since cardiac tissues are not easily accessible from patients, we explored whether cerium can be detected in the serum using neutron activation analysis (NAA). Cerium levels in serum of EMF patients were significantly elevated (p < 0.05) compared to controls.     

Proteome analysis of serum from type 2 diabetics with nephropathy

Diabetic nephropathy (DN) is a renal disease which develops as a consequence of diabetes mellitus. Microalbuminuria is the earliest clinical sign of DN. There are no specific diagnostic biomarkers for type 2 diabetics with nephropathy other than microalbuminuria and macroalbuminuria. However, microalbuminuria does not constitute a sole independent indicator for type 2 diabetics with nephropathy, and thus, another screening method, such as a biomarker assay, is required in order to diagnose it more correctly. Therefore, we have utilized two-dimensional electrophoresis (2-DE) to identify human serum protein markers for the more specific and accurate prediction of progressive nephropathy in type 2 diabetes patients, via comparisons of the serum proteome in three experimental groups: type 2 diabetes patients without microalbuminuria (DM, n = 30), with microalbuminuria (MA, n = 29), and with chronic renal failure (CRF, n = 31). As a result, proteins which were differentially expressed with statistical significance (p < 0.05) in MA and CRF groups as compared to those in DM group were selected and identified by ESI-Q-TOF MS/MS. Among these identified proteins, two proteins which might be useful as diagnostic biomarkers of type 2 diabetics with nephropathy were verified by Western blotting: extracellular glutathione peroxidase (eGPx) and apolipoprotein (ApoE) were found to exhibit a progressive reduction in MA and CRF groups. Notably, eGPx was further verified by ELISA using DM (n = 100) and MA (n = 96) patient samples. Collectively, our results show that the two proteins identified in this study may constitute potential biomarkers for the diagnosis of type 2 diabetics with nephropathy.     

Subnormal follicular-phase serum progesterone levels and elevated follicular-phase serum estradiol levels in young women with insulin-dependent diabetes

In a search for possible hormonal reasons for the loss of protection from myocardial infarction seen in diabetic women, serum levels of estradiol, progesterone, and luteinizing hormone were compared throughout a menstrual cycle (17 points) in eight healthy nonsmoking women and five otherwise healthy nonsmoking insulin-dependent diabetic women. The total length of the menstrual cycle and the lengths of the follicular and luteal phases did not differ between the groups. During the periovulatory and luteal phases, there was no significant intergroup difference with respect to any of the three hormones. During the follicular phase, in both groups, there was a plateau in serum progesterone concentration, with the level approximately 42% lower in the diabetic group (12.0 +/- 6.6 ng/dl versus 20.7 +/- 5.7; P less than 0.0001). Follicular-phase serum estradiol showed a rising curve in both groups; day-by-day comparison (days -10 to -3 before the luteinizing hormone peak) showed consistently higher levels in the diabetic group (mean, 108 pg/ml versus 95 pg/ml; P less than 0.001). The follicular-phase serum estradiol to progesterone ratio was nearly twice as high in the diabetic group as in the normal group (8.9 versus 4.6), a difference that was highly significant. The finding of elevated serum estradiol and subnormal serum progesterone concentrations during the follicular phase is so far unique to women with insulin-dependent diabetes mellitus. The possibility that this pronounced abnormality in diabetic women may be related to coronary disease merits testing in suitable in vivo and in vitro models of atherogenesis.     

Interpretation of elevated serum VEGF concentrations in patients with myocardial infarction

Our aim was to examine the relationship between the level of the inflammatory markers, C-reactive protein (CRP) and interleukin-6 (IL-6), and posttraumatic stress disorder (PTSD) symptomology in a random sample of 115 police officers. CRP was measured in citrated plasma using a particle enhanced immunonepholometric assay and IL-6 was measured in serum with a solid-phase quantitative sandwich ELISA. The presence of high PTSD symptomology was defined as having an Impact of Event Scale score (IES) of ≥ 26 compared to<26 (low PTSD symptomology). 28% of the officers had high PTSD symptomology. Mean levels of CRP and IL-6 did not differ significantly between officers with high PTSD symptomology and those with low symptomology (CRP: 0.76 mg/l vs. 0.97 mg/l; IL-6: 2.03 pg/ml vs. 1.74 pg/ml). We found no association of CRP and IL-6 levels with PTSD symptomology. This study was limited by sample size and its cross-sectional study design. A lack of association may occur if either CRP or IL-6 is elevated only at the onset of PTSD symptomology, or if inflammation is related to specific key components that define PTSD. Further research examining these relationships in a larger population may be worthwhile.     

MALDI-TOF/TOF-MS reveals elevated serum haptoglobin and amyloid A in Behcet's disease

Behcet's disease (BD) is a multisystemic autoimmune disease with unclear etiology and pathogenesis. To screen aberrant serum proteins in BD, serum samples were obtained from eight male BD patients with active uveitis and eight male healthy volunteers with informed consent. The serum samples from active BD patients and normal controls were pooled. Highly abundant serum proteins (albumin and IgG) were depleted from these two samples using an affinity capture based kit. The obtained samples were subjected to two-dimensional gel electrophoresis (2-DE). Protein spots were visualized with the "blue silver" staining. Differently expressed proteins were subsequently identified by matrix-assisted laser desorption /ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Western blot and enzyme-linked immunosorbent assay (ELISA) were performed using the serum samples from 18 patients with active BD, 6 patients with inactive BD, 22 patients with Vogt-Koyanagi-Harada (VKH) syndrome, and 20 healthy volunteers to validate the results of 2-DE and MS. Proteomic profiles of the pooled samples were compared, and approximately 800 protein spots were observed in each of the gels. Expression levels of four of the protein spots in active BD were significantly higher than those in the normal controls. Mass spectrometric protein identification revealed that the four protein spots corresponded to two proteins: haptoglobin (Hp) and serum amyloid A (SAA). Western blot and ELISA showed that Hp was only overexpressed in active BD but not in inactive BD, VKH syndrome, or healthy controls. An obvious band of SAA was detected in 72.2% of the serum samples from BD patients, whereas a vague band of this protein was found in 10.0% of the tested normal samples and 9.1% of VKH samples. Our results revealed a significantly increased expression of Hp and SAA in serum of active BD patients. These two proteins may be involved in the development of BD.     

Proteome analysis reveals adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung environment

Pseudomonas aeruginosa is known for the chronic lung colonization of cystic fibrosis (CF) patients in addition to eye, ear and urinary tract infections. With the underlying disease CF patients are predisposed to P. aeruginosa chronic lung infection, which leads to morbidity and mortality. In this study, we compared the protein expression profile of a CF lung-adapted P. aeruginosa strain C with that of the burn-wound isolate PAO. Differentially expressed proteins from the whole-cell, membrane, periplasmic as well as extracellular fraction were identified. The whole-cell proteome of strain C showed down-regulation of several proteins involved in amino acid metabolism, fatty acid metabolism, energy metabolism and adaptation leading to a highly distinct proteome pattern for strain C in comparison to PAO. Analysis of secreted proteins by strain C compared to PAO revealed differential expression of virulence factors under non-inducing conditions. The membrane proteome of strain C showed modulation of the expression of porins involved in nutrient and antibiotic influx. The proteome of the periplasmic space of strain C showed retention of elastase despite that the equal amounts were secreted by strain C and PAO. Altogether, our results elucidate adaptive strategies of P. aeruginosa towards the nutrient-rich CF lung habitat during the course of chronic colonization.     

Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology

Gametocytes, the precursor cells of malaria-parasite gametes, circulate in the blood and are responsible for transmission from host to mosquito vector. The individual proteomes of male and female gametocytes were analyzed using mass spectrometry, following separation by flow sorting of transgenic parasites expressing green fluorescent protein, in a sex-specific manner. Promoter tagging in transgenic parasites confirmed the designation of stage and sex specificity of the proteins. The male proteome contained 36% (236 of 650) male-specific and the female proteome 19% (101 of 541) female-specific proteins, but they share only 69 proteins, emphasizing the diverged features of the sexes. Of all the malaria life-cycle stages analyzed, the male gametocyte has the most distinct proteome, containing many proteins involved in flagellar-based motility and rapid genome replication. By identification of gender-specific protein kinases and phosphatases and using targeted gene disruption of two kinases, new sex-specific regulatory pathways were defined.     

Prognostic significance of elevated endothelin-1 levels in patients with colorectal cancer

BACKGROUND: Prognostic factors from clinical, laboratory and pathological data of patients with colorectal cancer are essential to identify high-risk groups to whom beneficial adjuvant therapy could be given. Endothelin-1, a growth factor, has been associated with the development and spread of solid tumours. This prospective study was performed to determine whether preoperative plasma big ET-1 levels might be useful as a prognostic indicator in patients with colorectal carcinoma. METHOD: Sixty-five consecutive patients with colorectal cancer confirmed by biopsy were included prospectively into this study over a 12-month period. Plasma samples from a peripheral vein were obtained prior to surgery. Univariate analysis of survival using age (< or > 70 years), sex, Dukes' stage (A&B versus C), tumour size (< or > 50 mm), vascular invasion and plasma big ET-1 levels was performed and significant factors were then analysed with the Cox regression model. RESULTS: Three variables, age, Dukes' tumour stage and plasma big ET-1 levels, were found to have prognostic significance (p<0.05). Factors associated with a poorer prognosis were age >70 years (p=0.02), Dukes' C tumours (p=0.04) and plasma big ET-1 levels >4.2 pg/mL (p=0.02). The Cox regression model identified the same three variables as having independent prognostic value for overall survival. CONCLUSION: Preoperative plasma big ET-1 levels may be useful in predicting overall survival in patients with colorectal cancer. Plasma big ET-1 levels may be useful in the selection of high-risk lymph node-negative patients with colorectal cancer for adjuvant therapy.     

Clonidine reduces elevated cerebrospinal fluid catecholamine levels in patients with essential hypertension

Cerebrospinal fluid (CSF) catecholamines were measured in normotensive patients and in patients with mild to moderate essential hypertension. CSF-norepinephrine (NE) concentrations were 50% lower in the normotensive individuals (127 ± 28 vs. 240 ± 23 pg/m1) (P<0.01). In hypertensive patients, CSF-NE was inversely related to age (r =-0.68; P<0.01) and directly related to plasma NE (r = 0.61; P<0.05). Clonidine (450 mcg/day for 2 weeks) significantly reduced CSF-NE (?40%) in hypertensive patients. In addition, it decreased blood pressure, plasma and urinary NE. Urinary VMA was not affected by clonidine. No correlation was observed between clonidine effects on BP and on plasma or CSF catecholamines. This study indicates that patients with essential hypertension have elevated levels of CSF-NE which are reduced after treatment with clonidine. The elevation of CSF-NE suggests that central (spinal?) noradrenergic activity may be increased in patients with mild to moderate essential hypertension, and that can be reduced by treatment with clonidine.     

Reduced serum acylated ghrelin levels in patients with hyperthyroidism

BACKGROUND AND OBJECTIVE: Recent studies have revealed that circulating ghrelin levels seem to play a role in energy homeostasis. The effect of hyperthyroidism on ghrelin levels is not fully known. METHODS: Serum levels of ghrelin and its relationship with insulin resistance were evaluated in 48 patients with hyperthyroidism and 43 euthyroid healthy controls. Thyroid hormones, insulin, glucose, ghrelin levels and lipid parameters were measured in all subjects. Insulin sensitivity was determined using the homeostasis model assessment. RESULTS: Serum ghrelin levels were significantly decreased in hyperthyroid patients than in controls (32.5 +/- 23.3 vs. 54.1 +/- 35.5 pg/ml, p < 0.001). Circulating ghrelin levels significantly correlated with age (r = -0.26, p = 0.01), fasting glucose (r = -0.21, p = 0.01), free triiodothyronine (r = -0.18, p = 0.04), free thyroxine (r = -0.23, p = 0.02) and thyroid stimulating hormone (r = 0.21, p = 0.04), but not with blood pressure, body mass index, lipid parameters, insulin and homeostasis model assessment (p > 0.05). Multiple regression analysis revealed glucose level to be the most important predictor of circulating ghrelin level. CONCLUSION: These results indicate that hyperthyroidism has effect on serum ghrelin levels. Further studies are needed for the exact mechanism.     

Differential expression of serum clusterin isoforms in colorectal cancer

Clusterin is an enigmatic protein altered in tumors of colorectal cancer patients. Because there is no information available about serum clusterin regarding this pathology, we applied proteomic techniques to analyze its isoforms in donors and patients. First we separated serum proteins through concanavalin A, obtaining a fraction with non- and O-glycosylated proteins (FI) and a second fraction enriched in N-glycoproteins (FII) wherein clusterin was supposed to elute on the basis of its glycosylation. Surprisingly analysis of the FI fraction revealed the existence of an unexpected and aberrantly N-glycosylated clusterin that was overexpressed in patients and comprised at least five isoforms with different isoelectric points. On the other hand, two-dimensional electrophoretic analysis of the clusterin eluted in FII detected one isoform that was increased and 15 isoforms that were decreased or absent in serum of patients. Finally immunoquantification by slot blot showed that in total serum and in FI the clusterin levels were significantly increased in patients, whereas in FII there was no significant variation. Therefore, serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.     

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.     

Increased levels of serum macrophage colony-stimulating factor before the onset of preeclampsia.

Macrophage colony-stimulating factor (M-CSF) is known to play a central role in maintaining pregnancy. The present study determined whether the increase in serum M-CSF levels preceded the onset of preeclampsia. Blood was collected from 110 normotensive pregnant women at risk for preeclampsia who were carrying single fetuses at about 30 weeks of gestation. After centrifugation, serum was stored at -20 degrees C until assay. Eighteen women developed preeclampsia at a later stage of pregnancy (group 1), while 88 women continued to have normotensive pregnancies until delivery. Thirty-four of the 88 women with normotensive pregnancy who were matched for age and parity were selected to form a control group (group 2). Serum M-CSF levels were determined by the sandwich ELISA method using three antibodies. Serum level of M-CSF was 1,266 U/ml (median) in group 1 and 1,082 U/ml in group 2. Serum M-CSF levels were significantly higher in group 1 than in group 2 (p < 0.0002). Increased levels of serum M-CSF markedly precede the development of clinical manifestations of preeclampsia. High serum M-CSF levels support M-CSF elevation in the placenta. This elevation at 30 weeks of gestation may be associated with placental hypoxia, which is considered the cause of preeclampsia.     

Proteome analysis reveals ubiquitin-conjugating enzymes to be a new family of interferon-alpha-regulated genes.

Interferon (IFN)-alpha is a cytokine with antiviral, antiproliferative, and immunomodulatory properties, the functions of which are mediated via IFN-induced protein products. We used metabolic labeling and two-dimensional gel electrophoresis followed by MS and database searches to identify potentially new IFN-alpha-induced proteins in human T cells. By this analysis, we showed that IFN-alpha induces the expression of ubiquitin cross-reactive protein (ISG15) and two ubiquitin-conjugating enzymes, UbcH5 and UbcH8. Northern-blot analysis showed that IFN-alpha rapidly enhances mRNA expression of UbcH5, UbcH6 and UbcH8 in T cells. In addition, these genes were induced in macrophages in response to IFN-alpha or IFN-gamma stimulation or influenza A or Sendai virus infections. Similarly, IFNs enhanced UbcH8 mRNA expression in A549 lung epithelial cells, HepG2 hepatoma cells, and NK-92 cells. Cycloheximide, a protein synthesis inhibitor, did not block IFN-induced upregulation of UbcH8 mRNA expression, suggesting that UbcH8 is the primary target gene for IFN-alpha and IFN-gamma. Ubiquitin conjugation is a rate-limiting step in antigen presentation and therefore the upregulation of UbcHs by IFNs may contribute to the enhanced antigen presentation by macrophages. Our results show that proteome analysis of cells is a suitable method for identifying previously unrecognized cytokine-inducible genes.     

Hemorrhage induces an increase in serum TNF which is not associated with elevated levels of endotoxin

Although tumor necrosis factor (TNF) and interleukin 6 (IL 6) are purported to be important mediators of inflammatory responses following trauma, it is not known if the serum levels of these cytokines are altered by simple hemorrhage. The objective of this study therefore was to determine whether or not: 1) there is any elevation of TNF or IL 6, and 2) if endotoxin, an important upregulator of these cytokines, is also increased following hemorrhage. To study this, C3H/HeN mice were bled to, and maintained at a mean blood pressure of 35 mmHg for 60 min, and then resuscitated with their own shed blood and adequate fluid. Mice were sacrificed at 30 min into hemorrhage and at 2, 4 or 24 hr post-hemorrhage to obtain serum samples. IL 6 and TNF levels were measured using cytokine dependent cellular assays. Using a quantitative Limulus amebocyte lysate assay, endotoxin levels were determined. TNF levels were significantly elevated at 30 min into hemorrhage, remaining so at 2 hr after resuscitation, but absent by 4 hr. Although there was a trend toward elevated IL 6 levels at 2 hr following hemorrhage, which was sustained up to 24 hr, the values were not significantly different from sham controls. When compared to controls, no marked increase in endotoxin was seen at any time point during or following hemorrhage. These results indicate that hemorrhage, in the absence of significant tissue trauma, causes enhanced TNF release which is not the result of increased endotoxin.     

Proteome analysis of vinca alkaloid response and resistance in acute lymphoblastic leukemia reveals novel cytoskeletal alterations

Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the beta-tubulin subunit of alpha/beta-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: beta-tubulin, alpha-tubulin, actin, heat shock protein 90beta, 14-3-3tau, 14-3-3epsilon, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance.