Elevated levels of mitochondrial mortalin and cytosolic HSP70 in blood as risk factors in patients with colorectal cancer.

Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70 (HSP70). It protects cells from senescence and apoptosis and is overexpressed in cancer cells. Cell resistance to complement-dependent cytotoxicity depends on mortalin and during complement attack mortalin is released from cells. Our goal was to determine whether cancer patients have circulating mortalin in blood. The significance of mortalin in blood to survival prospects of colorectal cancer patients was evaluated. Occurrence of extracellular soluble HSP70 (sHSP70) is documented. We developed a sensitive ELISA for mortalin. The association between mortalin level and survival was subjected to the Cox proportional hazards analysis (univariate and multivariate analyses). Mortalin concentration in serum of colorectal cancer patients was 10-214 ng/ml. Survival data of the patients were known from an earlier study of sHSP70 in these samples. Cox regression analysis indicated that high mortalin (>60 ng/ml) is a risk factor for shorter survival. Serum levels of sHSP70 and mortalin in patients were independent variables. Concurrence of high sHSP70 and mortalin was associated with rapid disease progression (HR = 4, 2.04-8.45, p < 0.001). Addition of high sHSP70 and mortalin to a baseline model of age, sex and TNM stage, significantly (p < 0.001) enhanced the risk score to 8 (3.26-20.46). This is the first demonstration of circulating mortalin in cancer patients. Analysis of mortalin in blood, and even more so of mortalin and sHSP70, adds a high prognostic value to the TNM stage and will identify colorectal cancer patients at high risk of poor survival.

ACTH-induced cortisol release is related to the copy number of the C4B gene encoding the fourth component of complement in patients with non-functional adrenal incidentaloma.

OBJECTIVE: According to our previous findings, carriers of the C4B*Q0 genotype, which means zero or one copy of the C4B gene, which is located in the RCCX copy number variation region on chromosome 6, have a significantly shorter life-expectancy and higher risk of cardiovascular disease than non-carriers. We have postulated that the C4B*Q0 genotype is linked to variant(s) of the neighboring CYP21A2 gene encoding a steroid 21-hydroxylase with altered function. DESIGN: Single-center, observational, retrospective study. PATIENTS: Seventy-six patients with non-functional, benign adrenal incidentaloma. MEASUREMENTS: Serum cortisol, aldosterone, 17-hydroxyprogesterone, corticosterone and ACTH levels basally and after ACTH-stimulation, metyrapone or dexamethasone tests were determined. C4B gene copy number was quantified. RESULTS: The ratio of ACTH-stimulated and baseline cortisol concentrations was significantly higher (P = 0·001) in the group of patients carrying the C4B*Q0 genotype compared to the rest of the patients. This difference remained significant (P = 0·004) after adjustment for sex and age, as well as for tumor size. A significant (P = 0·018), adjusted difference between carriers and non-carriers was found also for ACTH-induced/basal aldosterone ratio. In C4B*Q0 carriers, metyrapone hardly reduced the serum cortisol level, while in non-carriers it induced a highly significant (P = 0·002) decrease. CONCLUSIONS: The C4B*Q0 genotype may be associated with hyperreactivity of the HPA axis (manifested as an increased responsiveness to ACTH-stimulation), probably through enhanced function of steroid 21-hydroxylase. Since hyperreactivity of the HPA axis is known to be associated with an increased risk of cardiovascular disease, our present findings may explain the increased cardiovascular morbidity and mortality of C4B*Q0 carriers.

Parameters of the classical complement pathway predict disease severity in hereditary angioedema.

Functional C1-inhibitor (C1-inh) and C4 are potential severity markers of hereditary angioedema due to deficiency of C1-inh (HAE-C1-inh), and the complexes generated through complement activation may be relevant. We studied the association between disease severity and complement parameters in 105 HAE-C1-inh patients. Disease severity was characterized by the number of angioedema attacks or alternatively, by the number of C1-inh concentrate ampoules (C1-inh-amp) used for the treatment of attacks. Median C1rC1sC1-inh level was higher (32.8 U/ml vs. 3.4 U/ml; p<0.0001) in patients, compared to controls. C1rC1sC1-inh and C1-inh strongly correlated with attack number and C1-inh-amp, both in the whole patient population and in the subgroup on danazol prophylaxis. Both C1rC1sC1-inh and C1-inh are suitable for predicting disease severity based on attack frequency and C1-inh-amp (OR=4.38[1.43-13.43], p=0.010 and 11.78[2.54-54.67], p=0.002, respectively). We presume that both C1rC1sC1-inh and C1-inh might prove sensitive predictors of the severity of HAE-C1-inh.

Analysis of the 8.1 ancestral MHC haplotype in severe, pneumonia-related sepsis.

The most frequent Caucasian MHC haplotype, AH8.1 - associated with numerous immunopathological differences and certain autoimmune diseases - was recently linked to the delayed onset of bacterial colonization in cystic fibrosis. Based on this observation, we hypothesized that the carriers of AH8.1 have lower risk for a worse outcome in sepsis. AH8.1 carrier state was determined in 207 Caucasian patients with severe, pneumonia-related sepsis. Our data showed that in patients without chronic obstructive pulmonary disease (COPD), septic shock - a serious consequence of the bacterial infection - occurred significantly less frequently (OR=0.3383; 95% CI=0.1141-0.995; p=0.043) in carriers of AH8.1, than in non-carriers. According to the multivariate logistic regression analysis, this haplotype had an independent protective role against septic shock in all patients (OR=0.315; 95% CI=0.100-0.992; p=0.048), particularly in COPD-free patients (OR=0.117; 95% CI=0.025-0.554; p=0.007). These results indicate that AH8.1 may confer protection against the progression of bacterial infection, and this could explain, at least partially, its high frequency in the Caucasian population.

Lack of increased prevalence of immunoregulatory disorders in hereditary angioedema due to C1-inhibitor deficiency.

Hereditary angioedema due to deficiency of C1-INH (HAE-C1-INH) is associated with enhanced consumption of the early complement components, which may predispose for autoimmune disease. We assessed the prevalence of such disorders among HAE- C1-INH patients and their impact on the natural course of HAE-C1-INH. Clinical data and immunoserological parameters of 130 HAE-C1-INH and 174 non-C1-INH-deficient patients with angioedema were analyzed. In our study, the incidence of immunoregulatory disorders was 11.5% in the population of HAE-C1-INH patients and 5.2% among non-C1-INH-deficient controls with angioedema. Immunoserology screening revealed a greater prevalence of anticardiolipin IgM (p=0.0118) among HAE-C1-INH patients, than in those with non-C1-INH-deficient angioedema. We did not find higher prevalence of immunoregulatory disorders among our HAE-C1-INH patients. However, in patients with confirmed immunoregulatory disorders, the latter influenced both the severity of HAE-C1-INH and the effectiveness of its long-term management. Appropriate management of the immunoregulatory disease thus identified improves the symptoms of HAE-C1-INH.

Low ficolin-3 levels in early follow-up serum samples are associated with the severity and unfavorable outcome of acute ischemic stroke.

BACKGROUND: A number of data indicate that the lectin pathway of complement activation contributes to the pathophysiology of ischemic stroke. The lectin pathway may be triggered by the binding of mannose-binding lectin (MBL), ficolin-2 or ficolin-3 to different ligands. Although several papers demonstrated the significance of MBL in ischemic stroke, the role of ficolins has not been examined. METHODS: Sera were obtained within 12 hours after the onset of ischemic stroke (admission samples) and 3-4 days later (follow-up samples) from 65 patients. The control group comprised 100 healthy individuals and 135 patients with significant carotid stenosis (patient controls). The concentrations of ficolin-2 and ficolin-3, initiator molecules of the lectin complement pathway, were measured by ELISA methods. Concentration of C-reactive protein (CRP) was also determined by a particle-enhanced immunturbidimetric assay. RESULTS: Concentrations of both ficolin-2 and ficolin-3 were significantly (p < 0.001) decreased in both the admission and in the follow-up samples of patients with definite ischemic stroke as compared to healthy subjects. Concentrations of ficolin-2 and ficolin-3 were even higher in patient controls than in healthy subjects, indicating that the decreased levels in sera during the acute phase of stroke are related to the acute ischemic event. Ficolin-3 levels in the follow-up samples inversely correlated with the severity of stroke indicated by NIH scale on admission. In follow-up samples an inverse correlation was observed between ficolin-3 levels and concentration of S100ß, an indicator of the size of cerebral infarct. Patients with low ficolin-3 levels and high CRP levels in the follow up samples had a significantly worse outcome (adjusted ORs 5.6 and 3.9, respectively) as measured by the modified Rankin scale compared to patients with higher ficolin-3 and lower CRP concentrations. High CRP concentrations were similarly predictive for worse outcome, and the effects of low ficolin-3 and high CRP were independent. CONCLUSIONS: Our findings indicate that ficolin-mediated lectin pathways of complement activation contribute to the pathogenesis of ischemic stroke and may be additive to complement-independent inflammatory processes.

Long-term efficacy of danazol treatment in hereditary angioedema.

BACKGROUND: No systematic study has been published yet on the long-term efficacy of attenuated androgens in hereditary angioedema (HAE). Our aim was to conduct a follow-up study in two (German and Hungarian) cohorts of HAE patients (45 and 39 patients, respectively) undergoing uninterrupted treatment for 6 years with similar (starting dose 128 ± 78 mg per day and 136 ± 70 mg per day, respectively) and constant doses of danazol. DESIGN: The frequencies of subcutaneous, abdominal and laryngeal attacks were recorded each year. RESULTS: The annual frequency of all the three types of attacks was significantly lower during the first year of danazol treatment, compared to the last year before baseline. During subsequent years in Hungarian patients, the frequency of both subcutaneous and abdominal attacks - but not that of laryngeal attacks - increased significantly. In the case of abdominal attacks, a significant increase in the attack frequency was observed only in female patients. In the German cohort, by contrast, no change in the frequency of either type of attack was found during the 6-year study period. CONCLUSIONS: The differences observed between these cohorts cannot be related to drug dose, the age or gender distribution of subjects or the age at the onset of symptoms or the length of diagnostic delay in the patients. There were, however, marked differences in the baseline pattern of attacks: significantly - 3 times - more abdominal attacks were recorded in German patients. Further studies are necessary to clarify the mechanism of these findings.

New cholesterol-specific antibodies remodel HIV-1 target cells' surface and inhibit their in vitro virus production.

The importance of membrane rafts in HIV-1 infection is still in the focus of interest. Here, we report that new monoclonal anticholesterol IgG antibodies (ACHAs), recognizing clustered membrane cholesterol (e.g., in lipid rafts), rearrange the lateral molecular organization of HIV-1 receptors and coreceptors in the plasma membrane of HIV-1 permissive human T-cells and macrophages. This remodeling is accompanied with a substantial inhibition of their infection and HIV-1 production in vitro. ACHAs promote the association of CXCR4 with both CD4 and lipid rafts, consistent with the decreased lateral mobility of CXCR4, while Fab fragments of ACHAs do not show these effects. ACHAs do not directly mask the extracellular domains of either CD4 or CXCR4 nor do they affect CXCR4 internalization. No significant inhibition of HIV production is seen when the virus is preincubated with the antibodies prior to infection. Thus, we propose that the observed inhibition is mainly due to the membrane remodeling induced by cholesterol-specific antibodies on the target cells. This, in turn, may prevent the proper spatio-temporal juxtaposition of HIV-1 glycoproteins with CD4 and chemokine receptors, thus negatively interfering with virus attachment/entry.

Baseline level of functional C1-inhibitor correlates with disease severity scores in hereditary angioedema.

The diagnosis of hereditary angioedema (HAE) is based on complement tests. We studied for the first time the possible association between complement parameters measured at the time of diagnosis and disease severity in 115 patients with HAE. Serum levels of functional C1-inhibitor (C1-INH(f)), antigenic C1-inhibitor (C1-INH(a)), C4 and hemolytic activity of the classical pathway (CH50) were determined at the time of diagnosis. We found a significant correlation between severity scores and baseline C1-INH(f) levels, as determined by ELISA assay (p=0.0003). On the other hand, there was no correlation between severity scores and other complement parameters (C1-INH(a), C4, and CH50). We consider the correlation between severity scores and baseline C1-INH(f) levels an important pathophysiological observation. Our findings underlie the potential significance of monitoring functional C1-INH levels in relation to clinical disease course.

4G/5G polymorphism of PAI-1 gene is associated with multiple organ dysfunction and septic shock in pneumonia induced severe sepsis: prospective, observational, genetic study.

INTRODUCTION: Activation of inflammation and coagulation are closely related and mutually interdependent in sepsis. The acute-phase protein, plasminogen activator inhibitor-1 (PAI-1) is a key element in the inhibition of fibrinolysis. Elevated levels of PAI-1 have been related to worse outcome in pneumonia. We aimed to evaluate the effect of functionally relevant 4G/5G polymorphism of PAI-1 gene in pneumonia induced sepsis. METHODS: We enrolled 208 Caucasian patients with severe sepsis due to pneumonia admitted to an intensive care unit (ICU). Patients were followed up until ICU discharge or death. Clinical data were collected prospectively and the PAI-1 4G/5G polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism technique. Patients were stratified according to the occurrence of multiple organ dysfunction syndrome, septic shock or death. RESULTS: We found that carriers of the PAI-1 4G/4G and 4G/5G genotypes have a 2.74-fold higher risk for multiple organ dysfunction syndrome (odds ratio [OR] 95% confidence interval [CI] = 1.335 - 5.604; p = 0.006) and a 2.57-fold higher risk for septic shock (OR 95%CI = 1.180 - 5.615; p = 0.018) than 5G/5G carriers. The multivariate logistic regression analysis adjusted for independent predictors, such as age, nosocomial pneumonia and positive microbiological culture also supported that carriers of the 4G allele have a higher prevalence of multiple organ dysfunction syndrome (adjusted odds ratio [aOR] = 2.957; 95%CI = 1.306 -6.698; p = 0.009) and septic shock (aOR = 2.603; 95%CI = 1.137 - 5.959; p = 0.024). However, genotype and allele analyses have not shown any significant difference regarding mortality in models non-adjusted or adjusted for acute physiology and chronic health evaluation (APACHE) II. Patients bearing the 4G allele had higher disseminated intravascular coagulation (DIC) score at admission (p = 0.007) than 5G/5G carriers. Moreover, in 4G allele carriers the length of ICU stay of non-survivors was longer (p = 0.091), fewer ventilation-free days (p = 0.008) and days without septic shock (p = 0.095) were observed during the first 28 days. CONCLUSIONS: In Caucasian patients with severe sepsis due to pneumonia carriers of the 4G allele of PAI-1 polymorphism have higher risk for multiple organ dysfunction syndrome and septic shock and in agreement they showed more fulminant disease progression based on continuous clinical variables.