Detection of an antigen, associated with cells during the blast crisis of chronic myeloleukemia, in a cytotoxic test using xenogenic antibodies]

The immune antimyeloblast serum (AMS) was obtained from horses immunized with white blood cells from patients suffering from chronic myeloid leukemia (CML) at the blast crisis stage; the serum was completely absorbed with normal red blood cells and white blood cells (WBC). The absorbed antiserum remained cytotoxic to blast cells from 20 of 42 patients with CML at the blast crisis stage. AMS failed to react with the WBC from patients with CML in its chronic phase, and from patients with other types of leukemia Morphological studies indicated a possibility of identification of the antigen associated with myeloblasts from the blood of patients with CML blast crisis, by means to AMS.     

Successful interferon alpha therapy in atopic dermatitis of Besnier's prurigo pattern with normal serum IgE and blood eosinophil fraction: randomized case-controlled study

A randomized case-controlled study was carried out to investigate for interferon alpha therapy in atopic dermatitis (AD) of Besnier's prurigo pattern with normal serum IgE and normal blood eosinophil fraction. Interferon alpha therapy was conducted on 14 non-responders to interferon gamma and subsequent thymopentin therapy among 100 atopic dermatitis patients. Eight patients who improved significantly, showed skin lesions of the Besnier's prurigo pattern with normal serum IgE and normal blood eosinophil fraction. For the randomized prospective case-controlled study, 44 patients with the above characteristics were selected. Thirteen Besnier's prurigo patients were treated with interferon alpha therapy, ten with interferon gamma, ten with thymopentin, and the remaining 11 were untreated as the control group. With interferon alpha therapy, 11 out of 13 Besnier's prurigo patients with normal IgE and normal blood eosinophil fraction improved significantly, two out of ten improved with interferon gamma therapy, and none improved with thymopentin therapy or in the untreated control group. Interferon alpha therapy was effective on AD of Besnier's prurigo pattern with normal serum IgE and normal blood eosinophil fraction. These results suggest the possibility of non-IgE-mediated AD and the heterogeneity of atopic dermatitis.     

Anti-anaerobic activity of serum from patients treated with tigecycline for skin/soft tissue infections

To gain additional data concerning the anti-anaerobic activity of tigecycline in serum, we analyzed blood samples from six patients with a complicated skin/soft tissue infection who were receiving IV tigecycline 50 mg every 12 h. Venous blood samples were obtained after multiple doses of tigecycline at 1, 6 and 12 h after the initiation of a 1 h IV infusion. Sera from these samples were tested to determine serum inhibitory and bactericidal activity over time against 4 anaerobic bacteria (Bacteroides fragilis, Peptoniphilus asaccharolyticus, Prevotella bivia and Finegoldia magna). An analysis of serum titers found that tigecycline exhibited early (1 h) and prolonged (12 h) inhibitory activity against each study isolate. Moreover, it provided bactericidal activity for 12 h against these strains with the exception of F. magna. Tigecycline was found to exhibit antibacterial activity at serum concentrations below the MICs of the anaerobic bacteria tested. This finding further supports that the antimicrobial activity of tigecycline can be greater than that suggested by the free fraction of drug and that serum appears to enhance this antibacterial activity.     

Photoregulation of L-phenylalanine ammia-lyase: an immunochemical approach

The photoregulation of L-Phenylalanine ammonia-lyase (PAL) is studied by immunochemical methods. We used a partly purified L-Phenylalanine ammonia-lyase: F1 light fraction, the corresponding inactive one provided from dark-grown cotyledons: F1 dark fraction and the antisera specific of these two fractions. The complete absorption of PAL activity from F1 light fraction with the anti-F1 light immune serum shows the antigenicity of PAL and the specificity of this serum for all forms of PAL present in F1 light fraction. The presence of an inactive L-Phenylalanine ammonia-lyase in the 36 h dark-grown cotyledons suggested by preliminary results of absorption is conformed by showing that less PAL activity is precipitated from the fraction F1 light by a same amount of IgG anti-F1 light when F1 dark fraction is added. This result is explained by a competition between active and inactive forms of PAL for the IgG extracted from an immune serum specific for F1 light fraction. By measuring the absorption of PAL activity from FO fraction (crude extract) obtained from 18 h, 36 h and 48 h light-grown cotyledons when increasing amounts of IgG anti-F1 36 h light are added, we demonstrate the presence of at least two isozymes A and B, the synthesis of B being shifted in time in comparison to A.     

Role of biotin-binding affinity in streptavidin-based pretargeted radioimmunotherapy of lymphoma

One pretargeting approach to cancer radioimmunotherapy utilizes an antibody-streptavidin conjugate that is first localized to the tumor. A "clearing agent" is then administered to remove the excess bioconjugate from blood, followed by injection of the radiolabeled biotin therapeutic. In this study, the role of streptavidin-biotin affinity in this pretargeting system was investigated for the first time in vivo, with a reduced affinity, site-directed streptavidin mutant and with radiolabeled bis-biotin reagents. The S45A streptavidin mutant (SA-S45A), which displays a faster off-rate for biotin, was utilized with a bivalent biotin carrier that retains high avidity for the streptavidin mutant. Mice were fed either a normal or biotin-deficient diet, yielding serum endogenous biotin concentrations of 31 nM and 5 nM, respectively. Lymphoma-bearing nude mice pretargeted with 1F5 Antibody-SA-Wild Type (WT) bioconjugates produced (125)I-bis-biotin tumor concentrations of 2.2%ID/g and 7.0%ID/g in mice fed normal diets vs biotin-deficient diets. (125)I-bis-biotin tumor concentrations of mice pretargeted with 1F5-SA-S45A were 12%ID/g and 10%ID/g for mice fed normal and biotin-deficient diets, respectively. However, poor clearance of the 1F5-SA-S45A with the biotinylated clearing agent led to high normal organ concentrations of (125)I-bis-biotin. A galactosylated human serum albumin (HSA) modified with bis-biotin was then tested, and normal organ (125)I-bis-biotin concentrations were significantly reduced. Tumor-to-organ ratios achieved for 1F5-SA-S45A with the HSA-bis-biotin clearing agent in mice with high serum biotin were similar to those achieved with 1F5-SA-WT in mice with low serum biotin. These results demonstrate that exchange of bound endogenous biotin with lower affinity streptavidin mutants is possible, and that corresponding use of bis-biotin carriers can nearly eliminate the differences in therapeutic radioactivity at the tumor site in animals on normal vs biotin-deficient diets. The results also interestingly demonstrate, however, that improved clearance agents capable of removing the lower affinity streptavidin-antibody conjugate are needed to achieve comparable specificity in tumor to blood or normal organ ratios.     

Decreases in the serum VLDL-TG/non-VLDL-TG ratio from early stages of chronic hepatitis C: alterations in TG-rich lipoprotein levels

Background

The liver secretes very-low-density lipoproteins (VLDLs) and plays a key role in lipid metabolism. Plasma total triglyceride (TG) level variations have been studied in patients with hepatitis C virus (HCV)-related chronic hepatitis (CH-C). However, the results of these studies are variable. A homogenous assay protocol was recently proposed to directly measure the TG content in VLDL (VLDL-TG) and VLDL remnants.

Methodology/Principal Findings

Using the assay protocol, we determined serum VLDL-TG levels in 69 fasting patients with biopsy-proven HCV-related chronic liver disease and 50 healthy subjects. Patients were classified into stages F0–F4 using the 5-point Desmet scale. Serum total TG levels in patients with non-cirrhotic (F1–F3) CH-C did not demonstrate significant differences compared with healthy subjects, but serum VLDL-TG levels did demonstrate significant differences. Mean serum VLDL-TG levels tended to decrease with disease progression from F1 to F4 (cirrhosis). Compared with healthy subjects, serum non-VLDL-TG levels significantly increased in patients with stages F2 and F3 CH-C; however, we observed no significant difference in patients with liver cirrhosis. Furthermore, the serum VLDL-TG/non-VLDL-TG ratio, when taken, demonstrated a significant decrease in patients with CH-C from the mildest stage F1 onward.

Conclusions/Significance

The decrease in serum VLDL-TG levels was attenuated by increase in non-VLDL-TG levels in patients with non-cirrhotic CH-C, resulting in comparable total TG levels. Results of previous studies though variable, were confirmed to have a logical basis. The decrease in the serum VLDL-TG/non-VLDL-TG ratio as early as stage F1 demonstrated TG metabolic alterations in early stages of CH-C for the first time. The involvement of TG metabolism in CH-C pathogenesis has been established in experimental animals, while conventional TG measurements are generally considered as poor indicators of CH-C progression in clinical practice. The serum VLDL-TG/non-VLDL-TG ratio, which focuses on TG metabolic alterations, may be an early indicator of CH-C.     

Decreased rate of cytolysis of Colpidium striatum by cystic fibrosis serum. I. Bioassay and evidence for the possible involvement of a C/F factor--IgG complex

Treatment of the protozoan ciliate $\text{Colpidium}$$\text{striatum}$ with whole serum from cystic fibrosis (C/F) patients, their parents (obligate heterozygote carriers), and normal controls uniformly caused complete cytolysis of the protozoa. At a 1:10 dilution of serum, a distinct decrease in the rate of cytolysis was found for 16 out of 19 cystic fibrosis patients, and 12 out of 16 heterozygote samples. An investigation into the cause of the decreased cytolytic rate indicated that a serum component associated with the IgG fraction from homozygote and heterozygote serum was responsible for the decreased rate.     

Proteomic analysis of ovarian cancer proximal fluids: validation of elevated peroxiredoxin 1 in patient peripheral circulation

Background

Epithelial ovarian cancer (EOC) is the deadliest gynecologic malignancy in the United States. Unfortunately, a validated protein biomarker-screening test to detect early stage disease from peripheral blood has not yet been developed. The present investigation assesses the ability to identify tumor relevant proteins from ovarian cancer proximal fluids, including tissue interstitial fluid (TIF) and corresponding ascites, from patients with papillary serous EOC and translates these findings to targeted blood-based immunoassays.

Methodology/Principal Findings

Paired TIF and ascites collected from four papillary serous EOC patients at the time of surgery underwent immunodepletion, resolution by 1D gel electrophoresis and in-gel digestion for analysis by liquid chromatography-tandem mass spectrometry, which resulted in an aggregate identification of 569 and 171 proteins from TIF and ascites, respectively. Of these, peroxiredoxin I (PRDX1) was selected for validation in serum by ELISA and demonstrated to be present and significantly elevated (p = 0.0188) in 20 EOC patients with a mean level of 26.0 ng/mL (±9.27 SEM) as compared to 4.19 ng/mL (±2.58 SEM) from 16 patients with normal/benign ovarian pathology.

Conclusions/Significance

We have utilized a workflow for harvesting EOC-relevant proximal biofluids, including TIF and ascites, for proteomic analysis. Among the differentially abundant proteins identified from these proximal fluids, PRDX1 was demonstrated to be present in serum and shown by ELISA to be elevated by nearly 6-fold in papillary serous EOC patients relative to normal/benign patients. Our findings demonstrate the facile ability to discover potential EOC-relevant proteins in proximal fluids and confirm their presence in peripheral blood serum. In addition, our finding of elevated levels of PRDX1 in the serum of EOC patients versus normal/benign patients warrants further evaluation as a tumor specific biomarker for EOC.     

Analysis of PI (phosphatidylinositol)-anchoring antigens in a patient of paroxysmal nocturnal hemoglobinuria (PNH) reveals deficiency of 1F5 antigen (CD59), a new complement-regulatory factor.

FACS analysis together with PIPLC treatment was applied to PI-anchoring antigens such as DAF (decay-accerelating factor, CD55), 1F5 antigen (CD59), CD14 and CD16 on the cell surfaces of blood cells from a normal adult and a male patient with paroxysmal nocturnal hemoglubinuria (PNH). Through the extensive analysis, this patient proved to be completely defective in 1F5 antigen, a newly found complement-regulatory protein, on all the blood cells tested. In normal blood cells such as lymphocytes, monocytes and granulocytes, 1F5 antigen was expressed as one of PI-anchoring proteins. In contrast to most of PNH patients, this patient reserved DAF, CD14 and CD16 at normal levels in his erythrocytes, monocytes and granulocytes. Also, there were no significant differences between the normal adult and the patient in the activities of erythrocyte acetylcholinesterase and granulocyte alkaline phosphatase which were also known to be PI-anchoring enzymes. Thus, deficiency of 1F5 antigen must be deeply related to the clinical symptoms of PNH in this patient.     

General and local reactions in the proteolytic system during the development of experimental pneumonia

Proteolytic enzymes and their inhibitors were studied in the serum and bronchoalveolar lavage of rats with acute and chronic experimental pulmonary inflammation. An increase in the inhibitory potential of the blood serum and bronchoalveolar lavage at the acute stage of inflammation was shown. At the chronic stage of pulmonary inflammation the level of the main inhibitors decreased to normal or zero values in the blood and lavage fluid, respectively. It was observed that alpha 1-proteinase inhibitor serum concentration correlated with the level of bronchoalveolar antitrypsin.     

Triglycerides, fatty acids and cortisol in simple obesity in children

In 50 children with obesitas simplex, 6-14 years of age, the triglycerides (TG), free fatty acids (FFA) and cortisol (F) levels in venous blood serum were estimated. In agreement with the development stages, studied patients were divided into the group of younger children in prematurity stage and the group of older children approaching the maturity. Obtained mean values of TG, FFA and F concentrations were analysed in the particular groups of obese children and compared with the healthy children of the same age. Mean concentrations of TG, FFA and cortisol in obese children were within the normal values and statistically did not differ from those of control healthy children. Also there was no difference in parameters studied if compared the younger and older groups of obese children. There was no interrelationship between the high birth weight and the degree of overweight as well as between the duration of obesity and the blood serum TG levels. In the course of obesitas simplex in children no detectable disturbances in the levels of TG, FFA and cortisol were found. It may depends on the more efficient adaptational mechanism connected with metabolism which are acting in the course of overfeed in the period of growth and development.     

Elevation of an acid ribonuclease in serum of patients with chronic granulocytic leukaemia.

Ribonuclease (Ribonucleate nucleotide 2'-transferase E.C. 2.7.7.17) activity in serum of patients with chronic granulocytic leukaemia measured at pH 4.5-6.0 amounts to more than three times of that in serum of healthy subjects. At pH 6.0-8.0 the elevation of ribonuclease activity in serum of patients with chronic granulocytic leukaemia is less pronounced and amounts to about two times of that in normal ones. Using chromatography on CM Sephadex C-50 column, serum ribonuclease of both normal and chronic granulocytic leukaemia patients was separated into five distinct fractions. In serum of healthy subjects ribonuclease fractions denoted I-V contribute to 10; 21; 29; 22, and 18 percent of the total ribonuclease activity. In the serum of patients with chronic granulocytic leukaemia a decrease in ribonuclease fraction III to merely 17 percent and an increase in contribution of fraction IV to 32 percent of total ribonuclease activity could be observed. The comparison of each individual concentration of fraction in normal and leukaemia patients serum reveals, that ribonuclease fraction IV will increase about 3 times. A less pronounced increase could also be found for fractions I, II and V. However, ribonuclease fraction IV may be supposed to carry more than 50 percent of the whole extra load of ribonuclease present in the serum of chronic granulocytic leukaemia patients.     

Extract of gum resins of Boswellia serrata L. inhibits lipopolysaccharide induced nitric oxide production in rat macrophages along with hypolipidemic property

Boswellia serrata, Linn F (Burseraceae) is commonly used in Indian system of medicine (Ayurvedic) as an anti-inflammatory, analgesic, anti-arthritic and anti-proliferative agent. This study was planned to investigate the water-soluble fraction of the oleoresin gum of Boswellia serrata (BS extract) on lipopolysaccharide (LPS) induced nitric oxide (NO) production by macrophages under in vivo and in vitro conditions. In the previous condition, rats were fed on atherogenic diet (2.5% cholesterol, 1% cholic acid, 15.7 % saturated fat) along with the BS extract for 90 days. Blood was collected for lipid profile and toxicological safety parameters. Peritoneal macrophages were isolated and cultured to see the LPS induced NO production. Under in vivo experiment, BS extract significantly reduced serum total cholesterol (38-48 %), increased serum high-density lipoprotein- cholesterol (HDL-cholesterol, 22-30%). Under in vitro experiments with thioglycolate activated macrophages, it inhibited LPS induced (NO) production with IC 50 value at 662 ng /ml. Further, this fraction, in the dose of 15 mg/100 g body wt for 90 days, did not show any increase in serum glutamate-pyruvate transaminase (SGPT) and blood urea, in normal control animals. However, it significantly reversed the raised SGPT and blood urea in the atherogenic diet-fed animals. Transverse section of liver and kidney also supported its protective effect. Thus it may be concluded that water extract of Boswellia serrata possesses strong hypocholesterolemic property along with increase in serum HDL. It inhibits the LPS induced NO production by the activated rat peritoneal macrophages and show hepato-protective and reno-protective property.     

Effect of brain antibodies on the lipid peroxidation process in the brain

The authors studies the effects of blood serum and IgG fraction from dogs immunized with brain and blood sera from patients with multiple sclerosis and schizophrenia on lipid peroxidation in rat brain homogenates. Measured the content of diene conjugates (DC) and malonic dialdehyde (MDA) in the rat brain after administering the IgG fraction. It was established that antioxidant activity of blood sera and IgG fraction from control animals and donors was significantly higher as compared to experimental. Administration of the IgG fraction brought about an increase in the content of DC and MDA in the brain of experimental animals. It is concluded that complement-dependent brain antibodies present in the blood serum of patients with schizophrenia and multiple sclerosis potentiate lipid peroxidation in the cerebral tissue and that the unsophisticated and informative method for antibody determination may be used in clinical practice.     

Enzyme-linked immunosorbent assay in screening of leukemia-associated nuclear proteins

Our previous data revealed some diversities in electrophoretic characteristics of nuclear fraction proteins isolated from peripheral blood mononuclear cells of B-cell chronic lymphocytic leukemia (B-CLL) patients and healthy donors. Two electrophoretically-specific nuclear non-histones in the molecular mass zone of 38/39 and 44/46 kDa of leukemic mononuclear cells were used as immunogens to produce rabbit antisera. The Western blot analysis indicated that both nuclear components are expressed only in mononuclear cells isolated from peripheral blood of B-CLL patients, but not in those isolated from the blood of healthy donors. For further investigations of nuclear fraction from normal and B-CLL mononuclear cells, an enzyme-linked immunosorbent assay (ELISA) was used. The results obtained by ELISA with the antisera raised against both electrophoretically-specific B-CLL nuclear polypeptides revealed a different extend of cross-reactivity of nuclear fraction preparations isolated from normal cells and those isolated from leukemic ones. We noticed that nuclear fraction preparations which originated from leukemic mononuclear cells are much more reactive than normal ones with both antisera (at a broad range of antisera dilutions).     

Association of serum total bilirubin levels with diastolic dysfunction in heart failure with preserved ejection fraction

Background

Left ventricular diastolic dysfunction is one of the main characteristics of heart failure patients with a preserved left ventricular ejection fraction. As bilirubin is regarded as an important endogenous antioxidant molecule, serum total bilirubin levels were compared between heart failure patients with a preserved left ventricular ejection fraction and normal controls in this study. We recruited 327 heart failure patients with a preserved left ventricular ejection fraction and 200 healthy controls. Patients were divided into 4 subgroups by their comprehensive echocardiographic manifestations, 1-mild, 2-moderate, 3-severe (reversible restrictive), 4-severe (fixed restrictive). Total bilirubin levels were compared using stepwise multiple regressions adjusted for selected factors.

Results

After adjusting for gender, age, smoking, systolic blood pressure, diastolic blood pressure, total cholesterol and triglyceride, serum total bilirubin levels were significantly lower in the heart failure group compared with the control group (P < 0.01). Patients in the subgroup (4-severe) showed significantly (P < 0.05) lower levels of total bilirubin when compared with the subgroup (1-mild).

Conclusions

TB level was negatively correlated with left ventricular diastolic dysfunction in heart failure patients with a preserved left ventricular ejection fraction, which might provide a new insight into the complicated mechanisms of heart failure with a preserved left ventricular ejection fraction.     

Immunoenzyme determination of total serum ceruloplasmin. Application to Wilson disease

Enzyme immunoassay for ceruloplasmin (CP)*, employing monospecific CP antibodies labeled with horse radish peroxidase was developed. This method permits to determine total content of CP, which is present in Wilson disease patients' blood in enzymatically active and enzymatically inactive forms. The evidence is presented that the method can be used for a direct determination of CP in blood serum. The minimal CP concentration which may be determined by enzyme immunoassay (IEA) is 5.10(-9) g/ml. The method was used for determination of CP concentrations in Wilson disease patients' blood with different disease severity. Analysis of blood samples taken from 6 Wilson disease patients with the use of IEA method revealed similar total CP concentrations. At the same time, the oxidase activities of CP in the blood of different patients varied more than sevenfold.     

The role of foetal red blood cells in protecting cultured lymphocytes against diepoxybutane-induced chromosome breaks

Diepoxybutane (DEB) is an established mutagen that induces chromosome damage following in vitro treatment of peripheral blood lymphocytes. It is widely used to identify patients with Fanconi Anemia (FA), a clinical situation that is characterized, besides the hypersensitivity to DEB, by an elevated foetal haemoglobin (HbF) content in the peripheral blood. In a previous study, we showed that red blood cells (RBC) from normal individuals can protect cultured lymphocytes against chromosomal breaks induced by DEB and demonstrated the particular role of haemoglobin in the protective effect. In the present work, we studied the influence of RBC extracted from umbilical cord blood of neonates (F cells) on the frequency of DEB-induced chromosome breaks in lymphocyte cultures from normal individuals. Simultaneously, we determined individual GSTT1 and GSTM1 genotypes and the activity of Pi-class glutathione S-transferase (GSTP), catalase and superoxide dismutase (SOD) in adult and foetal RBC. Our results show that F cells, in comparison with adult RBC, elicit a better protection of cultured lymphocytes from normal individuals against chromosome breaks induced by DEB. Variability in the protective effect among RBC from different individuals was observed; we confirmed that the GSTT1 genotype modulates this inter-individual variability, but it is not sufficient to explain all of the protective effect of F cells. Our results suggest that the increased protective effect of F cells can be, at least in part, correlated with an increase in the activity of glutathione S-transferase, catalase and superoxide dismutase, in particular Cu/Zn SOD, in F cells compared with adult RBC.     

Increased fibroblast growth factor-like autoantibodies in serum from a subset of patients with cancer-associated hypercalcemia

Basic fibroblast growth factor (bFGF) is a potent tumor angiogenesis factor which lacks an amino-terminal signal sequence and does not normally circulate in serum from normal subjects. Naturally-occurring autoantibodies which mimicked basic fibroblast growth factor were described in serum from patients with multiple endocrine neoplasia type 1 prolactinoma or sporadic growth-hormone-secreting adenoma associated with increased bFGF. Since bFGF was increased in serum from a variety of cancers, we used endothelial cell proliferation assay(s) to test for bioactivity in the IgG fraction of serum from 56 patients with cancer-associated hypercalcemia, and normal or control subjects. We now report increased IgG-like endothelial cell activity in serum from a hyper prolactinemic subset (4/19 breast cancer; 1/14 renal cancer; 0/23 lung cancer) of cancer-associated hypercalcemic subjects. Highest activity was found in serum from three breast cancer patients who suffered spinal cord compression/metastases. The activity had properties of antiidiotype bFGF antibodies including reaction with anti-human IgG antibodies, and complete neutralization by rabbit antibodies to intact bFGF. The activity in endothelial cells persisted after storage at 0-4 C for 5 yrs; and [prepared by SDS-PAGE and immunoblotting with anti-human IgG] had apparent mol wt corresponding to the heavy chains of IgG. Serum IgG-like activity from 5 of 5 breast cancer patients and 2 of 2 prostate cancer subjects tested [prepared by anti-bFGF antibody, protein-A immunoaffinity, and hydroxyapatite (HA) chromatography] yielded peak HA-adsorbed activity that eluted with 0.4 M sodium phosphate, and was neutralized 70% by antibodies to intact bFGF. Cancer sera mean peak specific activity (12.0 ng-eq bFGF/ug protein) (n = 7) significantly exceeded (P < 0.001) normal sera mean peak specific activity (0.46 ng-eq bFGF/ug protein) (n = 6) in the 0.4 M sodium phosphate eluate fraction from hydroxyapatite columns. These results imply that long-lasting, bioactive FGF-like autoantibodies may arise spontaneously (and contribute to pathophysiology) in subsets of cancer patients with osseous metastases.