Towards retrievable vascularized bioartificial pancreas: induction and long-lasting stability of polymeric mesh implant vascularized with the help of acidic and basic fibroblast growth factors and hydrogel coating.

We seek to improve existing methodologies for allogenic grafting of pancreatic islets. The lack of success of encapsulated transplanted islets inside the peritoneal cavity is presently attributed to poor vascularization of the implant. A thick, fibrotic capsule often surrounds the graft, limiting survival. We have tested the hypothesis that neovascularization of the graft material can be induced by the addition of proper angiogenic factors embedded within a polymeric coat. Biocompatible and nonresorbable meshes coated with hydrophilic polymers were implanted in rats and harvested after 1-, 6-, and 12-week intervals. The implant response was assessed by histological observations on the degree of vascularity, fibrosis, and inflammation. Macrostructural geometry of meshes was conducive to tissue ingrowth into the interstitial space between the mesh filaments. Hydrogel coating with incorporated acidic or basic FGF in an electrostatic complex with polyelectrolytes and/or with heparin provided a sustained slow release of the angiogenic growth factor. Anti-factor VIII and anti-collagen type IV antibodies and a GSL I-B4 lectin were used to measure the extent of vascularization. Vigorous and persistent vascularization radiated several hundred microns from the implant. The level of vascularization should provide a sufficient diffusion of nutrients and oxygen to implanted islets. Based on our observations, stable vascularization may require a sustained angiogenic signal to allow for the development of a permanent implant structure.

Vascular endothelial growth factor effects on nuclear factor-kappaB activation in hematopoietic progenitor cells.

Vascular endothelial growth factor (VEGF) inhibits of the activation of transcription factor nuclear factor-kappaB (NF-kappaB) in hematopoietic progenitor cells (HPCs), and this is associated with alterations in the development of multiple lineages of hematopoietic cells and defective immune induction in tumor-bearing animals. Antibodies to VEGF have been shown to abrogate this effect. The mechanism by which VEGF antagonizes the induction of NF-kappaB was investigated in this study. Using supershift electrophoretic mobility shift analysis, we found that although tumor necrosis factor alpha (TNF-alpha) induced the nuclear translocation and DNA binding of p65-containing complexes, VEGF alone induced nuclear translocation and DNA binding of the complexes containing RelB. These results were confirmed by immunofluorescence confocal microscopy. VEGF effectively blocked TNF-alpha-induced NF-kappaB activation in HPCs from RelB-/- mice, however, similar to the effect observed in HPCs obtained from RelB+/- and RelB+/+ mice. This suggests that RelB is not required for VEGF to inhibit NF-kappaB activation. However, although TNF-alpha induced rapid activation of IkappaB kinase (IKK) as expected, this activity was substantially reduced in the presence of VEGF. This decreased IKK activation correlated with the inhibition of IkappaB alpha phosphorylation and degradation of IkappaB alpha and IkappaB epsilon in HPCs. VEGF alone, however, did not have any effect on phosphorylation of IkappaB alpha or degradation of IkappaB alpha and other inhibitory molecules IkappaB beta, IkappaB epsilon, or Bcl-3. SU5416, a potent inhibitor of the VEGF receptor I (VEGFR1) and VEGFR2 receptor tyrosine kinases, did not abolish the inhibitory effect of VEGF, indicating that the VEGF effect is mediated by a mechanism unrelated to VEGFR1 or VEGFR2 tyrosine kinase activity. Thus, VEGF appears to inhibit TNF-alpha-induced NF-kappaB activation by VEGFR kinase-independent inhibition of IKK. Therapeutic strategies aimed at overcoming VEGF-mediated defects in immune induction in tumor-bearing hosts will need to target this kinase-independent pathway.

A functional fibroblast growth factor-1 immunoglobulin fusion protein.

Proteins of the fibroblast growth factor (FGF) family play diverse roles in embryonic development, angiogenesis, and wound healing. The most well studied targets of FGF activity typically are cells of mesodermal and neuroectodermal origin; in addition, expression of FGF-1 (acidic FGF) is increased at several sites of chronic immunologic injury, and recent studies show that FGF-1 also may interact with cells of the immune system. In some human T cells, FGF-1 can induce signals necessary for production of interleukin-2, a key cytokine required for T cell proliferation. To better characterize the interaction of FGF-1 with FGF receptors on T cells, a fusion protein was constructed containing a portion of the constant region of human IgG1 (Fc) at the amino terminus of FGF-1. The Fc-FGF-1 fusion protein retained FGF function as determined by stimulation of tyrosine phosphorylation and DNA synthesis in NIH 3T3 cells. Binding of the intact fusion protein to FGF receptor 1 (FGFR1) on T cells was demonstrated by immunoprecipitation of the receptor bound to Fc-FGF-1 and by flow cytometry showing binding of fusion protein to T cells expressing FGFR1. This functional Fc-FGF-1 protein should prove useful in identifying FGFR-expressing cells.

Mast cell procarboxypeptidase A. Molecular modeling and biochemical characterization of its processing within secretory granules.

Previously, we characterized murine mast cell procarboxypeptidase A (MC-proCPA) as an inactive zymogen. To investigate the mechanisms for this lack of enzymatic activity and the processing of the zymogen to the active form, we now have performed molecular modeling of the tertiary structure of murine MC-proCPA based on the x-ray crystallographic structures of porcine pancreatic procarboxypeptidases A and B. Our model predicts that MC-proCPA retains a high degree of structural similarity to its pancreatic homologues. The globular propeptide physically blocks access to the fully formed active site of the catalytic domain and contains a salt bridge to the substrate-binding region that precludes docking of even small substrates. Based on consideration of the predicted tertiary structure and charge field characteristics of the model, the activation site (between GluA94 and Ile1) appears to be highly exposed even after MC-proCPA binds to secretory granule proteoglycans. Based on the steady-state levels of MC-proCPA versus MC-CPA, cycloheximide inhibition of protein synthesis, and brefeldin A blockage of protein sorting, we show that MC-proCPA is processed rapidly in murine mast cell line KiSV-MC14 with a half-life of 26 +/- 5 min (mean +/- S.D., n = 3), and the processing occurs within the secretory granules. The enzyme responsible for this processing may be a thiol protease since treatment of the KiSV-MC14 with 200 microM E-64d, a selective thiol-protease inhibitor, increases MC-proCPA by 2.7 +/- 0.2-fold (mean +/- S.D., n = 3) within 6 h of application.

Processing of procarboxypeptidase A and other zymogens in murine mast cells.

By cDNA sequence analyses the proteases found within the secretory granules of immune/inflammatory cells appear to be translated initially as zymogens, but by amino-terminal sequencing they are stored within the granules in an active form. We now show that murine mast cell carboxypeptidase A (MC-CPA) is produced in a zymogen form (MC-pro-CPA) that is present at approximately 0.5% of the level of MC-CPA. MC-pro-CPA is an inactive precursor of MC-CPA and is located within the secretory granules of the mast cells. We have identified one mast cell line, KiSV-MC9, that produces MC-pro-CPA yet cannot process it to the active form despite the fact that these cells can process prochymase and protryptase to their active forms, indicating that a separate mechanism exists for activation of the serine proteases. We show that dipeptidylpeptidase-I is involved in the processing of murine mast cell prochymase and procathepsin G, but does not process MC-pro-CPA or protryptase. Thus, mast cell carboxypeptidase, tryptase, and chymase zymogens are each processed to their active forms by different mechanisms.

[Fluoroimmunometric method of determining digitoxin]. / Fluoroimmunometricheskii metod opredeleniia digitoksina.

Digitoxin at concentrations up to 5 x 10(-10) M (therapeutic concentrations are 2 x 10(-8) M) can be reliable measured by a fluoroimmunometric method with coproporphyrin as a tracer. The use of nylon filters with immobilized digitoxin to remove an excess of labelled antibodies increases reliability and reduces the time of measurements, and thus simplifies the assay.

Evaluation of dissociation constants of antigen-antibody complexes by ELISA.

In this communication some of the advantages and constraints in the use of ELISA (enzyme-linked immunosorbent assay) procedures to evaluate antigen-antibody dissociation constants (Kd) are discussed and experimental conditions under which the effective Kd is close to the true value are proposed. Interactions between horseradish peroxidase (POD), human myoglobin and insulin with mono- and polyclonal antibodies (McAb and PcAb) were used to demonstrate that ELISA can be used to determine the average Kd, characterizing the interaction between antigens and PcAb. The Kd values obtained by ELISA were similar to those determined by luminescent immuno-cofactor analysis (LICA).

[Myoglobin and nickel in the blood of patients in the acute period of myocardial infarct]. / Mioglobin i nikel' v krovi bol'nykh v ostrom periode infarkta miokarda.

The content of blood nickel and myoglobin was studied in 25 patients with macrofocal and transmural myocardial infarction during the acute stage. It was established that acute myocardial infarction results in an increase of the content of nickel and myoglobin in the blood of these patients. The diagnostic importance of these examinations is discussed.

FN-C1q and C1 INH C1r-C1s complexes as indicators of complement activation in patients with chronic lymphocytic leukaemia.

We have previously found low levels of C1 and C4 INH in the sera of chronic lymphocytic leukaemia (CLL) patients. Hypocomplementaemia was supposed to be the consequence of a permanent activation of the classical pathway. We have compared the levels of C1 INH-C1rC1s and C1q-FN complexes in the sera of 95 CLL patients and 100 healthy controls, because these complexes are known to be formed in the early stage of classical pathway activation. A significant increase in the level of both types of complexes was found in sera of CLL patients as compared to the controls. These findings support the assumption that the classical complement pathway is activated in the patients with CLL.

[Comparative characterization of the polystyrene plates used in enzyme immunoassay]. / Sravnitel'naia kharakteristika polistirolovykh planshetov, primeniaemykh v IFA.

The data on the comparison of the quality of polystyrene plates manufactured in the USSR with those manufactured by a number of foreign producers are presented. These data indicate that the capacity of antibodies adsorbed on a polystyrene plate for the sorption of antigens depend on the properties of the polymer surface and vary for different plates.

[Dynamics of changes in the levels of myoglobin and anti-myoglobin autoantibodies in the blood serum of patients with myocardial infarction]. / Dinamika izmenenii kontsentratsii mioglobina i autoantitel k mioglobinu v syvorotke krovi bol'nykh s infarktom miokarda.

The mean level of myoglobin and autoantibodies to myoglobin in the blood of healthy donors was 77.57 +/- 8.17 ng/ml and 18.01 +/- 1.85 micrograms/ml respectively. The level of myoglobin in the blood of patients with primary transmural myocardial infarction was rapidly increased, reaching its maximum in 9-12 h and returning to normal in 9 days. The mean level of autoantibodies was decreased in the first 66 h and got back to normal by the 6th day of disease. In primary large focal nontransmural myocardial infarction the concentration of myoglobin in the blood of patients was also increased, reaching its maximum in 3-9 h and returning to normal by the end of the 2nd day after onset of an angina attack. A decrease in the level of autoantibodies to myoglobin was observed up to the 18th day of disease. The peculiarity of repeated large focal nontransmural myocardial infarction was a two-peak curve of changes in a MG level with maximum levels in 9-12 and 21-24 h after onset of a pain attack. Final normalization of the level of myoglobin in the blood of patients of this group occurred in 69 h. The concentration of autoantibodies to myoglobin was more than once decreased up to the 6th day of disease. The results obtained showed that groups of examinees differed in the time course of changes in the level of myoglobin and autoantibodies to myoglobin. Such differences can be used for diagnostic purposes.

[Fibrinogen and fibrin degradation products in the blood of acute myocardial infarct patients at the hospital rehabilitation stage]. / Produkty degradatsii fibrinogena i fibrina v krovi bol'nykh ostrym infarktom miokarda na étape bol'nichnoi reabilitatsii.

An enzyme immunoassay was used for a study of the time course of the content of fibrinogen degradation products (FDP) and free hemoglobin (fHg) in the blood of patients with acute myocardial infarction (AMI) during uncomplicated hospital rehabilitation. A considerable increase in the levels of FDP in the blood serum and fHg in the blood plasma of the AMI patients were noted. These levels were particularly high on the 6-12th day of rehabilitation with further fluctuations on the 22-24th day from the beginning of disease resulting from an increase of the patients' motor activity during rehabilitation which might cause the depletion of endothelial reserves of fibrinolysis activators and an increase in thrombinemia, however changes in the content of FDP and fHg in the blood were more likely associated with DIC-syndrome inherent fluctuations in the system of hemostasis. The content of FDP and fHg in the blood of AMI patients was recommended to be used as a marker of DIC-syndrome and assessment of corrective therapy.

[Ultramicromethod of erythrocyte immunoadsorption in screening for somatic antigens in acute myocardial infarct]. / Ul'tramikrometod éritroimmunoadsorbtsii v skrininge somaticheskikh antigenov pri ostrom infarkte miokarda.

Test-systems for the identification of somatic myoglobin antigens and fibrin-fibrinogen degradation products in the sera of patients with acute myocardial infarction have been developed and tested. A significant correlation between erythrocyte immunoadsorption technique and solid-phase immunoenzyme assay was observed, the former technique being simpler and express.

[Possibilities of using immunoenzyme test systems for determining levels of myoglobin, fibrinogen, fibrin-fibrinogen degradation products and fibronectin in the diagnosis of somatic diseases]. / Vozmozhnosti primeneniia immunofermentnykh test-sistem dlia opredeleniia soderzhaniia mioglobina, fibrinogena i produktov degradatsii fibrin-fibrinogena, fibronektina v diagnostike somaticheskikh zabolevanii.

Clinical trials of immunoenzyme test-systems for the quantitative determination of fibronectin, fibrinogen, fibrin/fibrinogen degradation products and myoglobin have been performed on serum and plasma samples obtained from patients and healthy donors. The tests were informative and possessed diagnostic value in the following conditions: fibronectin--in pyogenic septic complications of newborns, burn infections; fibrinogen and fibrin/fibrinogen products--in thrombosis, myocardial infarction and disseminated intravascular coagulation syndrome; myoglobin--in myocardial infarction.

[Determination of binding constants of antibodies fractionated according to their affinity to insulin]. / Opredelenie konstant sviazyvaniia antitel, fraktsionirovannykh po srodstvu k insulinu.

The number of populations of antibodies differing in their affinity for insulin was determined in guinea pig antiserum. The antibody fractions differing in their affinity were obtained by affinity chromatography on an antigenic sorbent. Elution with a stepwise pH gradient from 4 to 2 differing in the number and form of the steps resulted in 10 fractions of antibodies obtained from individual animal sera. The binding constants of fractionated antibodies were determined by a bioluminescent immunocofactor method. With a change in elution pH from 4 to 2, the equilibrious binding constant of antibodies increased from 10(6) to 5 X 10(9) M-1.

[Solid-phase immunoenzyme method of determining myoglobin in the diagnosis of acute myocardial infarction]. / Tverdofaznyi immunofermentnyi metod opredeleniia mioglobina v diagnostike ostrogo infarkta miokarda.

A solid-phase immunoassay was developed for quantitative estimation of myoglobin in human biological fluids. This technique was used for analyses of blood serum and plasma from patients with acute myocardium infarction. The method developed was similar to radioimmunoassay by its diagnostic characteristics but more simple and available. The immunoassay might be used in diagnosis of myocardium infarction as well as in estimation of the disease severity at least in clinics.

[Determining myoglobinemia in acute myocardial infarction by the immunoenzyme method]. / Opredelenie mioglobinemii pri ostrom infarkte miokarda immunofermentnym metodom.

Like radioimmunoassay, immunoenzyme assay permits detecting myoglobinemia both in healthy persons and in patients with acute myocardial infarction (AMI). In AMI patients, myoglobinemia reaches a maximum 5 to 12 hours since the onset of a powerful anginal attack whereupon it gradually and spasmodically decreases, without returning to normal toward outcome of the 3d day of the observation period. A single assay of myoglobinemia in AMI patients is of diagnostic importance within 2 to 27 hours since the disease onset.