Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.     

The Changes of Lipid Metabolism in Advanced Renal Cell Carcinoma Patients Treated with Everolimus: A New Pharmacodynamic Marker?

Background

Everolimus is a mammalian target of rapamycin (mTOR) inhibitor approved for the treatment of metastatic renal cell carcinoma (mRCC). We aimed to assess the association between the baseline values and treatmentrelated modifications of total serum cholesterol (C), triglycerides (T), body mass index (BMI), fasting blood glucose level (FBG) and blood pressure (BP) levels and the outcome of patients treated with everolimus for mRCC.

Methods

177 patients were included in this retrospective analysis. Time to progression (TTP), clinical benefit (CB) and overall survival (OS) were evaluated.

Results

Basal BMI was significantly higher in patients who experienced a CB (p=0,0145). C,T and C+T raises were significantly associated with baseline BMI (p=0.0412, 0.0283 and 0.0001). Median TTP was significantly longer in patients with T raise compared to patients without T (10 vs 6, p=0.030), C (8 vs 5, p=0.042) and C+T raise (10.9 vs 5.0, p=0.003). At the multivariate analysis, only C+T increase was associated with improved TTP (p=0.005). T raise (21.0 vs 14.0, p=0.002) and C+T increase (21.0 vs 14.0, p=0.006) were correlated with improved OS but were not significant at multivariate analysis.

Conclusion

C+T raise is an early predictor for everolimus efficacy for patients with mRCC.     

Antibodies against gangliosides and ganglioside complexes in Guillain–Barré syndrome: New aspects of research

Guillain–Barré syndrome (GBS) is an acute autoimmune neuropathy, often preceded by an infection. Serum anti-ganglioside antibodies are frequently elevated in titer. Those antibodies are useful for diagnosis. Some of them also may be directly involved in the pathogenetic mechanisms by binding to the regions where the respective target ganglioside is specifically localized. We have recently found the presence of the antibody that specifically recognizes a new conformational epitope formed by two gangliosides (ganglioside complex) in the acute-phase sera of some GBS patients. In particular, the antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. Some patients with Miller Fisher syndrome also have antibodies against ganglioside complexes including GQ1b; such as GQ1b/GM1 and GQ1b/GD1a. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which the carbohydrate portions of two different gangliosides may form a new conformational epitope. Within the rafts, gangliosides are considered to interact with important receptors or signal transducers. The antibodies against ganglioside complexes may therefore directly cause nerve conduction failure and severe disability in GBS. More study is needed to elucidate the roles of the antibodies against ganglioside complexes.     

Prognostic Potential of the Panfungal Marker (1 → 3)-β-d-Glucan in Invasive Mycoses Patients

Mycopathologia - We analyze the prognostic potential of (1?→?3)-β-d-glucan (BG) levels in predicting clinical outcomes in patients with invasive fungal infections, on a...     

Sub-Inhibitory Concentrations of Human α-defensin Potentiate Neutralizing Antibodies against HIV-1 gp41 Pre-Hairpin Intermediates in the Presence of Serum

Human defensins are at the forefront of the host responses to HIV and other pathogens in mucosal tissues. However, their ability to inactivate HIV in the bloodstream has been questioned due to the antagonistic effect of serum. In this study, we have examined the effect of sub-inhibitory concentrations of human α-defensin HNP-1 on the kinetics of early steps of fusion between HIV-1 and target cells in the presence of serum. Direct measurements of HIV-cell fusion using an enzymatic assay revealed that, in spite of the modest effect on the extent of fusion, HNP-1 prolonged the exposure of functionally important transitional epitopes of HIV-1 gp41 on the cell surface. The increased lifetime of gp41 intermediates in the presence of defensin was caused by a delay in the post-coreceptor binding steps of HIV-1 entry that correlated with the marked enhancement of the virus'' sensitivity to neutralizing anti-gp41 antibodies. By contrast, the activity of antibodies to gp120 was not affected. HNP-1 appeared to specifically potentiate antibodies and peptides targeting the first heptad repeat domain of gp41, while its effect on inhibitors and antibodies to other gp41 domains was less prominent. Sub-inhibitory concentrations of HNP-1 also promoted inhibition of HIV-1 entry into peripheral blood mononuclear cells by antibodies and, more importantly, by HIV-1 immune serum. Our findings demonstrate that: (i) sub-inhibitory doses of HNP-1 potently enhance the activity of a number of anti-gp41 antibodies and peptide inhibitors, apparently by prolonging the lifetime of gp41 intermediates; and (ii) the efficiency of HIV-1 fusion inhibitors and neutralizing antibodies is kinetically restricted. This study thus reveals an important role of α-defensin in enhancing adaptive immune responses to HIV-1 infection and suggests future strategies to augment these responses.     

Serum Bile Acids in Repaired Tetralogy of Fallot: A Marker for Liver and Heart?

Background and Aims

Patients with repaired tetralogy of Fallot may develop chronic right ventricular dysfunction and hepatic congestion over time. We hypothesized that bile acid metabolism is altered in repaired tetralogy of Fallot patients and therefore sought to correlate right ventricular indices with serum bile acid levels.

Methods

Indexed right ventricular end diastolic volume, as assessed by cardiac magnetic-resonance imaging, was classified as <100ml/m² (Group 1, n = 5), 100–150ml/m² (Group 2, n = 18), and >150ml/m² (Group 3, n = 6) in 29 patients with repaired tetralogy of Fallot. Pulmonary regurgitation fraction and right ventricular ejection fraction were calculated. The serum bile acid profile, including 15 species, in these patients was determined by liquid chromatography coupled with mass spectrometry.

Results

Serum bile acid levels increased from Group 1 to Group 3 (2.5 ± 0.7; 4.1 ± 2.5; 6.0 ± 2.8 μmol/l, respectively) with significantly increased bile acid values in Group 3 compared to Group 1 (p≤0.05). In Group 3, but not in Group 1 and 2, a significant increase in glycine-conjugated bile acids was observed. Pulmonary regurgitation fraction increased (12 ± 1; 28 ± 16; 43 ± 3%, Groups 1–3, respectively) and right ventricular ejection fraction decreased (48.4 ± 6.4; 48.5 ± 6.5; 42.1 ± 5.3%, Groups 1–3, respectively) with rising indexed right ventricular end diastolic volume.

Conclusions

These preliminary results suggest that serum bile acid levels are positively correlated with indexed right ventricular end-diastolic volume in patients with repaired tetralogy of Fallot; however, this needs to be confirmed in a larger patient cohort.     

Screening for Distress in Routine Oncological Care—A Survey in 520 Melanoma Patients

Introduction

Despite the increasing incidence of melanoma little is known about patients'' emotional distress associated with this disease. Supplemented by the problem list (PL), the distress thermometer (DT) is a recommended screening instrument to measure psychosocial distress in cancer patients. Our objective was to explore the acceptance and the feasibility of the DT and PL as a concise screening tool in an ambulatory setting for routine care and to elucidate determinants of distress in melanoma patients with regard to sociodemographic and clinical variables.

Methods

Consecutive melanoma outpatients were asked to complete the DT with the PL prior to their scheduled consultation. Demographic and clinical data were obtained from the patients'' charts. Clinical data included melanoma stage, time since diagnosis, previous treatment, current treatment, and other cancer disease.

Results

Out of 734 patients recruited into the study, 520 patients (71%) completed both the DT and the PL. Forty-seven percent met the ≥5 cut-off score for distress. Younger and employed patients reported higher distress than older and retired patients. A cut-off score of ≥5 was closely associated with self-reported emotional sources of distress, with practical problems, especially at work, family problems (dealing with the partner), and physical problems like pain, appearance, getting around, and nausea. Apart from higher distress under current systemic treatment, no associations were found between distress and clinical data.

Conclusion

The DT together with the PL seems to be an economically reasonable screening tool to measure psychosocial distress in melanoma patients. In particular, younger melanoma patients who are currently employed are prone to experience distress at some point after diagnosis, but there appears to be almost no association between clinical data and the extent of distress. To characterize the impact of distress on disease outcome and quality of life in melanoma patients, further research is needed.     

Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78–13.2) compared to 8.26 months (95% CI 6.02–19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn’t reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.     

Serum S100B: A Potential Biomarker for Suicidality in Adolescents?

Background

Studies have shown that patients suffering from depression or schizophrenia often have immunological alterations that can be detected in the blood. Others reported a possible link between inflammation, a microgliosis and the blood-brain barrier (BBB) in suicidal patients. Serum S100B is a marker of BBB function commonly used to study cerebrovascular wall function.

Methods

We measured levels of S100B in serum of 40 adolescents with acute psychosis, 24 adolescents with mood disorders and 20 healthy controls. Patients were diagnosed according to DSM-IV TR criteria. We evaluated suicidal ideation using the suicidality subscale of the Brief Psychiatric Rating Scale for Children (BPRS-C).

Results

Serum S100B levels were significantly higher (p<0.05) and correlated to severity of suicidal ideation in patients with psychosis or mood disorders, independent of psychiatric diagnosis. Patients with a BPRS-C suicidality subscores of 1–4 (low suicidality) had mean serum S100B values +/− SEM of 0.152+/−0.020 ng/mL (n = 34) compared to those with BPRS-C suicidality subscores of 5–7 (high suicidality) with a mean of 0.354+/−0.044 ng/mL (n = 30). This difference was statistically significant (p<0.05).

Conclusion

Our data support the use of S100B as an adjunctive biomarker to assess suicidal risk in patients with mood disorders or schizophrenia.     

Detection of RASSF1A and RAR? Hypermethylation in Serum DNA from Breast Cancer Patients

Breast cancer is fast emerging as the leading cancer amongst females, especially in young females in metropolitan cities in India. The epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prognosis of the disease. Furthermore, using body fluids such as serum offers a non-invasive method to procure multiple samples for such analyses. In this study, we examined methylation status of two normally unmethylated but biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A) and Retionic acid receptor ? (RAR?) by Methylation Specific PCR (MSP) in invasive ductal carcinomas of the breast and paired serum DNA. RASSF1A was found to be methylated in 17 of 20 (85%) breast tumors; while sera from 15 of 20 (75%) of the patients showed concordant methylated RASSF1A, with a sensitivity of 88%. RAR? was methylated in 2/20 (10%) breast tumors. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA for both RASSF1A and RAR? genes; hence specificity was 100%. Immunohistochemical analysis of RAR? protein in 15 breast carcinoma patients harboring unmethylated RAR? in tumors and serum DNA showed the expression of RAR? protein in tumors and paired normal breast tissues, confirming the MSP findings, suggesting that RAR? promoter is functional in these cases. This study underscores the potential utility of DNA methylation based screening of serum, a readily accessible body fluid, as a surrogate marker for early detection of breast cancer.      

5-LOX in Alzheimer’s Disease: Potential Serum Marker and In Vitro Evidences for Rescue of Neurotoxicity by Its Inhibitor YWCS

The inflammatory process plays a key role in neurodegenerative disorder. The inflammatory molecule, 5-lipooxygenase (5-LOX), protein is involved in the pathologic phenotype of Alzheimer’s disease (AD) which includes Aβ amyloid deposition and tau hyperphosphorylation. This study determined the level of 5-LOX in serum of AD patients, mild cognitive impairment (MCI) patients, and the normal elderly, and the rescue effect by YWCS, a peptide inhibitor of 5-LOX on neurotoxicity by Aβ amyloid_25–35 (Aβ_25–35) in neuroblastoma cells. The concentration of serum 5-LOX was estimated by surface plasmon resonance and western blot. The neuroprotective effect of 5-LOX peptide inhibitor YWCS in Aβ_25–35-induced neurotoxicity was analyzed by MTT assay and western blotting. We found significant upregulated serum 5-LOX in AD patients and also in MCI patients compared to the normal control group. The peptide inhibitor of 5-LOX, YWCS, prevented the neurotoxic effect of Aβ_25–35 by reducing the expression of γ-secretase as well as p-Tau₁₈₁ in SH-SY5Y cells. However, YWCS was nontoxic towards normal HEK cells. The differential expression of serum 5-LOX among the study groups suggests it can be one of potential serum protein marker and a therapeutic regimen for AD and MCI. The negative correlation with neuropsychological parameters, i.e., MoCA and HMSE, increases its importance and makes it useful during the clinical setup which is very needful in developing countries. Peptide YWCS can serve as a new platform as a 5-LOX inhibitor which can prevent neurotoxicity developed in AD.     

Potential of thidiazuron in improved micropropagation of Picrorhiza kurroa– an endangered medicinal herb of alpine Himalaya

The potential of thidiazuron (1-phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea (TDZ) in the micropropagation of Picrorhiza kurroa, a western Himalayan herb was studied. The roots and rhizomes of this plant are rich in medicinally important glycosides i.e., picroside I, picroside II and kutkoside. Nodal segments (2.0–2.5 cm) from plants were pretreated with 0, 0.25, 0.50, 0.75, 1.0 μM TDZ for 7, 15 and 30 days. Maximum shoot multiplication was recorded after 60 days, provided, the nodal segments pretreated with 0.5 μM TDZ for 15 days were transferred to 0.8% agar gelled MS medium containing 3.0% sucrose. The shoots also showed profuse rooting having maximum length. When these plantlets were incubated at 15°C for 10 days and transferred to sand under polyhouse conditions, 100% survival was recorded after one month. In contrast, when the plantlets were not incubated at 15°C, only 86% survival was recorded. While the stomata and chlorophyll content of the tissue culture-raised plantlets treated at 15°C were comparable to that of plants growing in the field, the ones that were not treated at 15°C showed lower number of stomata and chlorophyll content.     

High Potential of a Transposon mPing as a Marker System in japonica �� japonica Cross in Rice

Although quantitative traits loci (QTL) analysis has been widely performed to isolate agronomically important genes, it has been difficult to obtain molecular markers between individuals with similar phenotypes (assortative mating). Recently, the miniature inverted-repeat transposable element mPing was shown to be active in the japonica strain Gimbozu EG4 where it had accumulated more than 1000 copies. In contrast, most other japonicas, including Nipponbare, have 50 or fewer mPing insertions in their genome. In this study we have exploited the polymorphism of mPing insertion sites to generate 150 PCR markers in a cross between the closely related japonicas, Nipponbare × Gimbozu (EG4). These new markers were distributed in genic regions of the whole genome and showed significantly higher polymorphism (150 of 183) than all other molecular markers tested including short sequence repeat markers (46 of 661). In addition, we performed QTL analysis with these markers using recombinant inbred lines derived from Nipponbare × Gimbozu EG4, and successfully mapped a locus involved in heading date on the short arm of chromosome 6. Moreover, we could easily map two novel loci involved in the culm length on the short arms of chromosomes 3 and 10.Key words: Linkage mapping, Transposon, japonica, Oryza sativa L., QTL analysis     

Sirtuin1: A Promising Serum Protein Marker for Early Detection of Alzheimer’s Disease

Sirtuin (SIRT) pathway has a crucial role in Alzheimer’s disease (AD). The present study evaluated the alterations in serum sirtuin1 (SIRT1) concentration in healthy individuals (young and old) and patients with AD and mild cognitive impairment (MCI). Blood samples were collected from 40 AD and 9 MCI patients as cases and 22 young healthy adults and 22 healthy elderly individuals as controls. Serum SIRT1 was estimated by Surface Plasmon Resonance (SPR), Western Blot and Enzyme Linked Immunosorbent Assay (ELISA). A significant (p<0.0001) decline in SIRT1 concentration was observed in patients with AD (2.27±0.46 ng/µl) and MCI (3.64±0.15 ng/µl) compared to healthy elderly individuals (4.82±0.4 ng/µl). The serum SIRT1 concentration in healthy elderly was also significantly lower (p<0.0001) compared to young healthy controls (8.16±0.87 ng/µl). This study, first of its kind, has demonstrated, decline in serum concentration of SIRT1 in healthy individuals as they age. In patients with AD and MCI the decline was even more pronounced, which provides an opportunity to develop this protein as a predictive marker of AD in early stages with suitable cut off values.     

Elevated Levels of IFN-γ in CSF and Serum of Patients with Amyotrophic Lateral Sclerosis

Objectives

To explore whether the levels of IFN-γ in cerebral spinal fluid (CSF) and serum are elevated in ALS patients and to analyze the correlations between the IFN-γ levels and disease progression.

Methods

CSF and serum samples were obtained from 52 ALS patients and 31 non-ALS patients. The levels of IFN-γ in CSF and serum were assessed, and disease progression parameters, including the disease interval (months from onset, MFO), the revised ALS Functional Rating Scale (ALSFRS-r) score and the disease progression rate (DPR) were analyzed by registered neurologists. All samples were measured using a commercial enzyme-linked immunosorbent assay. Statistical analyses were performed using Prism software.

Results

Compared to the non-ALS patients, the ALS patients displayed significantly increased levels of IFN-γ in both CSF and serum, and these values consistently correlated with disease progression.

Conclusions

These results demonstrated that IFN-γ in CSF may serve as a biomarker of ALS differentiation and progression. CSF IFN-γ was a more reliable biomarker of disease diagnosis and progression than serum IFN-γ.     

A2G Is a New System of α-2-Globulin Allotypes in Pig Blood Serum

Combinations of four -2-globulin allotypes were studied for their distribution in pigs of nine different breeds and hybrid groups. Based on this analysis, a new, previously unpublished polyallelic genetic system designated A2G was postulated. The complex alleles of this system control -2-globulin allotypes and are suggested to be encoded by genes of two subloci. One of these subloci is virtually monomorphic, whereas the other has at least four allelic variants.     

Antibodies against gangliosides and ganglioside complexes in Guillain-Barré syndrome: new aspects of research

Guillain-Barré syndrome (GBS) is an acute autoimmune neuropathy, often preceded by an infection. Serum anti-ganglioside antibodies are frequently elevated in titer. Those antibodies are useful for diagnosis. Some of them also may be directly involved in the pathogenetic mechanisms by binding to the regions where the respective target ganglioside is specifically localized. We have recently found the presence of the antibody that specifically recognizes a new conformational epitope formed by two gangliosides (ganglioside complex) in the acute-phase sera of some GBS patients. In particular, the antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. Some patients with Miller Fisher syndrome also have antibodies against ganglioside complexes including GQ1b; such as GQ1b/GM1 and GQ1b/GD1a. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which the carbohydrate portions of two different gangliosides may form a new conformational epitope. Within the rafts, gangliosides are considered to interact with important receptors or signal transducers. The antibodies against ganglioside complexes may therefore directly cause nerve conduction failure and severe disability in GBS. More study is needed to elucidate the roles of the antibodies against ganglioside complexes.