Elevated serum gamma globulins in apparently healthy Nigerians living in Ogbomoso: A possible manifestation of phagocytic dysfunction

Background: Serum protein electrophoresis abnormalities, particularly elevated gamma globulins (hypergammaglobulinemia), have been reported in apparently healthy Nigerians living in Ogbomoso and elsewhere. Since the mechanisms for this phenomenon have not been fully substantiated, we hypothesized that impaired neutrophil phagocytosis could contribute to this condition. Methods: Healthy humans exhibiting hypergammaglobulinemia (HGG) were identified using serum protein electrophoresis (SPE) performed on cellulose acetate gel in barbital buffer (pH 8.6). GelQuant image analysis and quantitation software were further employed to quantify gamma globulin fraction. Neutrophils were isolated from K3EDTA anticoagulated peripheral blood using neutrophil isolation histopaque of Kayman Chemical, USA. Neutrophil phagocytic activity was analyzed using a non-subjective commercial colorimetric phagocytosis assay kit obtained from Cell-Biolab Inc, USA. Results: The purity and viability of isolated neutrophils were approximately 94 % and 92 %, respectively. Ex-vivo phagocytic activity of neutrophils isolated from apparently healthy subjects exhibiting HGG, expressed in absorbance unit (AU), was 48.1±8.6 % which was significantly lower (p<0.05); compared to the controls (98.9±14.3 %). Conclusion: Since neutrophils play crucial roles in innate immune responses, impairment of neutrophil phagocytic activity may lead to persistent antigenic stimulations of the adaptive immune system. This could in turn orchestrate γ-globulins expression leading to HGG. Statement of novelty: We demonstrated a reduced neutrophil phagocytic activity as a possible basis for hypergammaglobulinemia in healthy Nigerians, perhaps for the first time.

PURIFICAÇÃO E ISOENZIMAS DA LACTATO DESIDROGENASE DO MÚSCULO EPAXIAL DE Prochilodus scropha e Notothenia neglecta

Foi levado a efeito um estudo comparativo de purificação da lactico desidrogenase (L-lactato NAD+ oxidorreductase, E.C.1.1.1.27) do músuclo epaxial do peixe tropical Prochilodus scropha e do peixe antártico Notothenia neglecta. A purificação da LDH de ambas as fontes foi procedida por cromatografia de afinidade em oxamato-agarose, tendo revelado uma única banda de proteína em eletroforese em acetato de celulose, para ambas as preparações. Contudo, foi verificado que em eletroforese em tampão barbital pH 8,6, a LDH de P. scropha migra para o cátodo enquanto que a de N. neglecta migra para o ânodo. Abstract It has been carried out a comparative study on the purification of the lactate dehydrogenase (Llactate NAD+ oxidoreductase, E.C.1.1.1.27) from the epaxial muscle of the tropical fish Prochilodus scropha and the Antarctic fish Notothenia neglecta. Purification of LDH from both sources was carried out by oxamateagarose affinity chromatography. The eletrophorectic profile in cellulose acetate showed a single band in both preparations. However it has been observed that in barbital buffer pH 8.6, the LDH from P. scropha migrates towards the cathode while the LDH from N. neglecta, migrates towards the anode.

Clinical Associations of an Increased Transthyretin Band in Routine Serum and Urine Protein Electrophoresis

The 9697 electrophoretograms performed over an 8 year period were reviewed to identify the frequency and clinical associations of the finding of a prominent transthyretin band in serum or urine, the concentration of which was equal to or greater than a 64 mg/dL protein calibrator. All samples were electrophoresed at a constant 90 V using agarose gels with a barbital buffer pH 8·6 and Ponceau S staining. Reference calibrators were used as standards to identify increased transthyretin bands and the patients' clinical records were subsequently reviewed. High values were found in 46 patients' sera and a further nine patients' urines representing 0·57% of the total workload. Renal impairment was present in 58% of cases including those with chronic renal failure, the nephrotic syndrome and paraproteinaemia. The high levels were not persistent in three myeloma cases where there was a recovery in renal function following chemotherapy. In some nephrotics, a high urine transthyretin may be secondary to a general hepatic albumin and transport protein synthesis response to severe proteinuria. Why the serum transthyretin was elevated in many other cases remains unclear.

Thyroxin binding by human serum albumin after denaturation of the thyroxin-binding globulin in familial dysalbuminemic hyperthyroxinemia.

Abnormal binding of thyroxin (T4) to serum albumin of subjects with familial dysalbuminemic hyperthyroxinemia (FDH) is generally demonstrated by the T4-loaded charcoal uptake test, with T4 added in excess (0.1 mmol/L) to accentuate T4 binding to albumin in FDH. I describe a binding study involving T4 tracer in which thyroxin-binding globulin is denatured in samples by treatment with mild acid at pH less than 3.0. The tracer is bound to the serum albumin and, to a greater extent, to the FDH albumin, because the binding by thyroxin-binding prealbumin is blocked by barbital buffer. The result of the [125I]T4 binding to the albumin is expressed as a T4 binding index, based on results for pooled sera from patients with normal thyroid function as a reference. The mean index in FDH was 4.08 (SD 0.92, n = 5); in hypoalbuminemia, 0.66 (SD 0.18, n = 8); in normal subjects, 1.00 (SD 0.11, n = 20). This albumin-binding index enables the rapid and unequivocal diagnosis of subjects with FDH, without the addition of unlabeled T4.

IMMUNOAFFINITY PURIFICATION OF PLASMA INHIBITOR FOR ACTIVATED PROTEIN C

Activated protein C (APC) is an important regulator of blood coagulation in vivo. In plasma this serine protease is slowly inhibited by a specific inhibitor, activated protein C inhibitor (PCI), (Suzuki et al. (1983) J.Biol.Chem. 258 , 163-168). We have now made monoclonal antibodies against PCI by immunizing with the APC-PCI complex. Positive clones were identified by solid phase immunoassay with 125I labelled partially purified inhibitor. After subcloning and expansion in mice, one of the monoclonal antibodies was immobilized on Sepharose 4B and used in the purification of the inhibitor. A two step purification procedure was deviced starting with passage of fresh human plasma over the column. Following extensive washing the inhibitor was eluted with 50 mM triethylamine- HCI,0.5 M NaCl, pH 11.0, from the column together with a small amount of high molecular weight material. After gel filtration on a column packed with AcA 44 the inhibitor appeared homogenous on SDS - PAGE. Approximatly 0.5 mg inhibitor was obtained from 200 ml of fresh plasma. The apparent Mr of the inhibitor was 57000 kDa on SDS -PAGE. The purified protein formed a complex (Mr =110000 kDa) with human APC. At the same time a band (Mr = 54000 kDa ) appeared that represented the modified inhibitor. When analyzed on agarose gel electrophoresis (75mM barbital buffer, 2mM EDTA at pH 8.7 ) the PCI migrated to the β2- region, whereas the modified inhibitor had a slightly more anodal mobility. The APC-PCI komplex migrated to the α2- region.Two immunoradiometric assays were constructed with the monoclonal antibodies. One measured the complexes between APC and PCI while the other one measured the total amount of PCI present. These assays were used to study complex formation in buffer and plasma.

PURIFICATION AND CHARACTERIZATION OF TWO PROCOAGULANTS FROM WALKER 256 CARCINOSARCOMA TUMORS,

Activation of the coagulation system bytumor cells may play an important role in tumor growth and metastases. Becauseprocoagulant activities have been identified in different tumor cells by different investigators, effective comparison of these activities has been difficult. Therefore, we purified and characterized two different procoagulant proteins from the same Walker 256 tumors. The first procoagulant activity/platelet aggregating activity (PCA/PAA) was purified from a 1% CHAPS detergent extract oftumor homogenate followed by (NH4)2SO4 fractionation, anion exchange and hydrophobic chromatography. The protein had a molecular weight of 58,000, required phospholipid and an intact coagulation pathway from factor X through fibrinogen for activity, but did not require factors VII or IX forits procoagulant activity. The procoagulant activity was not inhibited by 5mMphenyl-methyl sulfonyl fluoride, iodoacetamide or phenanthroline; there was noevidence of proteinase activity. The PAA was due to thrombin generation during coagulation. The second procoagulant,cancer procoagulant (CP), was extracted from tumors in barbital buffer (pH 7.4) without detergent, purified by immunoaffinity (using a polyclonal goat antibody to CP from V2 carcinoma) and mercurial-benzoate affinity chromatography. CP had a molecular weight of 68,000, an isoelectric point of 4.8 and initiated coagulation by directly activating factor X in the coagulation system. CP was inhibited by Hg++ and iodoacetamide, cysteine proteinase inhibitors. The purified CP formed an immunodiffusion precipitin band against the polyclonal anti-CP goat antibody. Thus, thepurified CP had the same physicochemical, enzymatic and immunologic propertiesas CP from rabbit V2 carcinoma. Neither procoagulant had the properties of tissue factor. These results suggest that there aretwo distinct procoagulant activities inWalker 256 and that both may contributeto the coagulation abnormalities that are associated with tumor growthand metastases.

Binding characteristics of thyroxin binding globulin in serum of normal, pregnant, and severely ill euthyroid patients.

Serum from normal, pregnant, and severely ill patients was stripped of endogenous thyroid hormones and diluted 1100-fold in barbital buffer. We then used it to study the binding characteristics of thyroxin binding globulin (TBG), noting significant differences in binding capacities among the groups. The mean (+/- SD) triiodothyronine/thyroxin ratio for binding capacity was 18 +/- 4 for normal subjects. The ratio was significantly increased in pregnant patients, 21 +/- 4 (p less than 0.05), and significantly lower in severely ill patients, 12 +/- 4 (p less than 0.05). When serum was diluted before assay, to give a uniform TBG concentration among groups, these apparent differences in binding characteristics were eliminated. It therefore is unlikely that different molecular species of TBG account for the variations in binding characteristics in these clinical states. Apparently, the distribution of thyroxin and triiodothyronine among the binding sites on TBG changes with variations in TBG concentration. This may explain the discrepancies observed in the concentrations of free thyroid hormones as estimated by various methodologies.

Assessment Of FVIII Functional Activities With Quantitative Electrophoresis

Electrophoretically separated fractions of FVIII concentrates were quantitatively assayed to assess heterogeneity in FVIII related proteins. The Method was successfully applied to the characterisation of multiple forms of FVIII in normal, pathological, haemophilia A, and v. WD. post-infused samples. Similarly, FVIII:C inactivation properties of several FVIII antibodies, which differed from one another on the basis on their complex formations, were investigated by: i) identifying the molecular forms of the Residual FVIII and ii) by monitoring the electrophoretic distribution of the complexes using a radiolabelled VIII:C IgG. 200 ul samples were applied to rectangular wells (22 × 3mm) cut in a 2.5mm thick agarosegel (1-2%). Electrophoresis in the first dimension was performed in 0.023M barbital buffer (pH8.2). The gels were then cut into 20-30 equal slices, eluted with 0.025M Tris-saline buffer (pH7.4) and assayed by currently used methods.The results indicate that FVIII concentrates are highly heterogeneous, consisting of several subpopulations with differing functional and inhibitor neutralization activities. Most classical assays (including IRMA) were sensitive to the larger forms but methods based on the optimal Fxa generations (VIII:C 2 stage & VIII:C amidolytic) measured smaller forms of FVIII more sensitively. In the post-infused samples a transient complex was observed which reflected the rate of clearance of FVIII from the circulation. Furthermore the novo synthesis of FVIII in v. WD patients appeared to be quantitatively indistinguishable from circulating FVIII present in these patients. Some populations of FVIII were totally and specifically inactivated by some FVIII:C inhibitors but partially by the others. The inhibitor which inactivate totally FVIII:Cam also failed to produce high molecular weight radiolabelled complexes. On the basis of these results, inhibitor patients could be treated specifically with an appropriate subpopulation of FVIII.

Molecular Weight of Human Plasma Factor VIII.

Recently it has been postulated that native plasma factor VIII is of low molecular weight. We have investigated the molecular weight of VIII: C by agarose chromatography. Factor VIII: C was eluting at the void volume under the following conditions. 1) Blood anticoagulated with 10mM benzamidine 10mM ε-aminocaproic acid and diisopropyl-fluoro-phosphate. Gelchromatography was performed with 10mM benzamidine, 10mM ε-aminocaproic acid and huuman albumin 40 mg/ml) in Michaelisbuffer at 37° in the presence or absence of 2mM Ca2+. 2) Heparin plasma 4U/ml) and elution in barbital buffer, 3) citrated plasma in michaelisbuffer, 4 As (3). but after prevention of disulfide bridge formation by collection of the blood into 5mM iodoacetic acid, 10mM N-ethylmaleimide or ImM p-chloromercuriphenylsulphonic acid. Similar elution patterns were obtained with factor VIII purified from cryoprecipitate. Upon reduction the molecular weight of both purified and plasma factor VIII decreased and the lowest molecular weight form with measurable coagulant activity was about 480.000. Incubation at high ionic strength (0.25M CaCl2) resulted in dissociation and the activity was associated with a molecular weight of 50 - 100.000. Gel filtration at pH 6.0 also resuited in retardation of factor VIII on agarose columns. The data suggests that the in vivo molecular weight of factor VIII is 1-2x106 and that alterations are due to changes in the in vitro conditions.

A non-barbital buffer for immunoelectrophoresis and zone electrophoresis in agarose gels.

We describe buffer for both immunoelectrophoresis and zone electrophoresis procedures in agarose gels, Tricine [N-tris(hydroxymethyl)methylglycine] and Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol] being the main components. This buffer eliminates the procurement cost and inventory control associated with formulation of barbital buffers. We compare it with other common buffer formulations.