Physical fitness and nutritional status of polish ground force unit recruits.

The purpose of the work was to conduct an examination of the physical fitness and nutritional status of recruits (221 men beginning military service in the infantry unit). Soldiers' physical efficiency was estimated using 4 tests: standing long jump, pull-ups on bar, 30-second sit-ups and 1000-metre run. The nutritional status assessment was done based on anthropometric measurements including measurements of body height, body mass and selected skin fold thickness. The study group of soldiers were the best at sit-ups (46.33 points). They got over 40 points for the 1000-metre run (43.68 points) and for pull-ups on bar (41.69 points). They obtained the lowest scores for standing long jumps (30.77 points). About 14% of recruits were overweight and 4.1% underweight. Recruits enrolling in the infantry unit present a low physical fitness level. Overweight and obesity occurrence, and particularly underweight, in recruits testify to improper nutrition before beginning military service.

The human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes.

The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin. It is unclear whether neutrophils also express the C3aR, but need a costimulus provided by eosinophils for certain biological responses, or whether neutrophils lack the C3aR and respond to C3a via a secondary stimulus generated by eosinophils, i.e., by an indirect mode. In the present study, polyclonal antiserum raised against the second extracellular loop of the C3aR was used to characterize C3aR expression on peripheral blood leukocytes. For high degree purification of neutrophils, a negative selection method was established that decreased the contamination with CD9(bright+) eosinophils down to <0.2%. Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling. Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood. The expression of the C3aR on eosinophils could be confirmed. In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).

Urban wastelands: On the frontline between air pollution sources and residential areas.

In urban areas, particulate matter (PM) represents an increasing threat to human health. The ability of plants in parks and along roads in cities to accumulate PM has already been demonstrated, but nothing is known about the effect of wasteland vegetation on air quality, despite a significant proportion of greenery in polluted areas being on wastelands. The aim of this study was to document the accumulation of PM and trace elements (TE) by wasteland species (Robinia pseudoacacia L., Populus × canescens (Aiton) Sm., Acer negundo L., Solidago gigantea (Aiton) and Poaceae) growing on Central European urban wastelands with differing levels of air pollution. On average, the largest amounts of PM accumulated on the foliage of R. pseudoacacia and S. gigantea, and the smallest amounts accumulated on P. × canescens leaves. However, accumulation of PM depended more on the distance from the emission source than on species selection, and was higher on the polluted wasteland where the plants' gas exchange was the lowest. The results also suggest that in order to effectively accumulate PM from the air, it is critical to have the correct configuration of plants, with the wasteland vegetation having a layered structure and layers differing in PM retention, as shown in this study using the examples of R. pseudoacacia (a tall tree with low PM retention) and S. gigantea (below-tree vegetation with high PM retention). P. × canescens accumulated the highest concentrations of Cd and Zn, S. gigantea accumulated the highest concentration of Cu, and Poaceae accumulated the highest concentrations of Cr and Ni. These findings have implications for urban vegetation management in areas where there is no organised greenery, and offer proof that vegetation in wasteland areas should be maintained since it is an excellent tool for reducing concentrations of PM at its place of origin.

Influence of folic acid, vitamin B2 and B6 supplementation on feed intake, body and organs weight, and liver fatty acids composition in rats subjected to severe protein deprivation.

Growing rats fed for 3 months a low-protein (LP) diet (4.5% of energy from protein), possessed about 29% lower body weight than animals consuming adequate-protein diet (20% energy from protein). The LP diet feeding caused an increase in daily feed intake followed by a decrease in feed conversion efficiency. The enrichment of LP diet with folic acid, vitamin B2 and B6 (3 times above the level applied in the control diet) did not have any impact on rats BW and supplementation with these vitamins minimize the effect of LP diet on feed intake. The use of examined vitamins had a tendency to diminish an increase in feed conversion ratio caused by the LP nutrition. This effect was significant when all vitamins were added together. Rats fed the LP diet had higher relative weights of lungs, heart, liver and testis. Vitamins enriching the LP diet were observed to decrease a relative weight of lungs (folic acid, vitamin B6 and vitamin mixture), and liver (vitamin B6 and vitamin mixture). A tendency of increasing relative testis weight was also revealed in rats given the LP diet enriched with vitamins. The lower content of hepatic polyunsaturated fatty acids (FA) and a tendency for monounsaturated FA content to be higher were found in rats fed the LP diet. The LP diet enrichment with folic acid caused that these changes were more pronounced and statistically significant. Enrichment of LP diet with vitamins tested may cause a partial reverse of changes observed in the hepatic FA composition.

Analysis of the C5a anaphylatoxin core domain using a C5a phage library selected on differentiated U937 cells.

The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35-40 was randomly mutated and the resulting C5a[35-40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A35E36R37A38S39R40], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q35E36R37I38L39N40] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.

A selection system to study C5a-C5a-receptor interactions: phage display of a novel C5a anaphylatoxin, Fos-C5aAla27.

Binding and effector domains of the human anaphylatoxin C5a have been determined by either site directed mutagenesis or synthetic peptide studies. However, the lack of specific selection methods, which allow direct investigation of C5a-C5a-receptor interaction made these studies laborious. To overcome these limitations we have constructed a novel Fos-C5a expressed on the tip of a filamentous phage. To guarantee for a free C-terminus which is required for C5a activity C5a cDNA was cloned into the phagemid vector pJuFo. Helper phage infection of pJuFc-C5a transformed cells resulted in a mutant phage displaying Fos-C5a on its surface. However studies with Bt2cAMP differentiated U937 cells revealed that phage displayed Fos-C5a is functional inactive. Subsequently we replaced a nonconserved cysteine residue at position 27 by alanine and obtained Fos-C5aAla27. Both the purified and the phage displayed Fos-C5aAla27 proteins were functional active and induced enzyme release from differentiated U937 cells. In addition, purified Fos-C5aAla27 exhibited the same binding profile as compared to rhC5a. Fos-C5aAla27 displaying phages were mixed with phage harboring only the pJuFo plasmid at a ratio of 10(6). After four successive rounds of panning on differentiated U937 cells Fos-C5aAla27 phages were enriched to 100% as shown by C5a-specific ELISA. We expect this approach to prove helpful for studying C5a-C5a-receptor interactions. i.e. to screen C5a libraries for high affinity binders with agonistic or antagonistic properties directly on cells.

G alpha-16 complements the signal transduction cascade of chemotactic receptors for complement factor C5a (C5a-R) and N-formylated peptides (fMLF-R) in Xenopus laevis oocytes: G alpha-16 couples to chemotactic receptors in Xenopus oocytes.

The human leukocyte chemoattractant receptors for complement factor C5a (C5a-R) and N-formylated peptides (fMLF-R) are important members of the superfamily of G-protein coupled receptors (GPCR). Uniquely among the GPCR, these two receptors cannot be expressed in a functionally active form in the oocytes of the frog Xenopus laevis, but require substitution of total RNA of the myelomonocytic U-937 or HL-60 cell lines, respectively. Recently, it was reported that the C5a-R may couple to the alpha subunit of G-16. We have tested this G-protein for its ability to complement the signal transduction cascade of the C5a-R and fMLF-R in Xenopus oocytes. Injection of cRNA for the C5a-R in combination with G alpha-16 led to expression of a functional C5a-R as measured by ligand-induced whole cell current. In contrast to a previous report, the fMLF-R exhibited some residual functional activity when transiently expressed in Xenopus oocytes the extent of which could, however, substantially be increased by coexpression of G alpha-16. Thus, G alpha-16 complements the signal transduction cascade of both receptors in Xenopus laevis oocytes and is most likely the complementing factor present in the U-937 and HL-60 cell lines.

Amino acids 327-350 of the human C5a-receptor are not essential for [125I]C5a binding in COS cells and signal transduction in Xenopus oocytes.

The anaphylatoxic peptide C5a is an important inflammatory mediator of the complement system. We have generated human C5a-receptor (hC5aR) mutants with truncation of its cytosolic carboxyl-terminus (C-terminus). Both mutants were analysed for C5a-binding in transiently expressing COS cells, and one mutant additionally for GTP-binding regulatory protein (G-protein) coupling in cRNA-injected Xenopus oocytes. Our data suggest that (a) amino acids (aa) 314 to 326 as part of the C-terminus are necessary for proper receptor folding or expression and (b) the receptor C-terminus distal from position 327 is not critical for receptor expression, folding, binding and G-protein coupling.

Functional expression of a human C5a receptor clone in Xenopus oocytes requires additional RNA.

cRNA from a PCR-generated C5aR clone was prepared by in vitro transcription and microinjected into Xenopus laevis oocytes. Ligand-induced whole cell current could be detected after co-injection of cRNA for the C5aR with total RNA of the unstimulated U937 cell line, but not with either of the components injected alone. These data clearly demonstrate an absolute requirement of the C5aR for an additional human factor to become functionally expressed in Xenopus oocytes.

Re-evaluation of the storage conditions for blood samples which are used for determination of complement activation.

EDTA-blood samples derived either from healthy staff or septic patients were investigated for in vitro complement activation during the first 48 h after blood drawing at 4 degrees C and 20 degrees C. For this purpose C3a/C3a desArg plasma levels were determined by the ABICAP C3a assay. Within the septic group no complement activation was detectable during the whole observation period. However, if blood from healthy persons was stored for longer than 6 h at 20 degrees C complement activation occurred. The most profound activation was found in EDTA-blood stored for 48 h at 20 degrees C. C3a values in this sample increased four-fold from 56 +/- 7 ng/ml to 222 +/- 38 ng/ml. From these data we conclude that both immediate cooling of EDTA-blood to 4 degrees C, as well as the immediate separation of plasma as proposed by Mollnes et al. (Clin. Exp. Immunol. (1988) 73, 484), is not necessary for determination of anaphylatoxin plasma values. Storage of EDTA-blood samples for up to 6 h without the need to perform centrifugation should allow anaphylatoxin measurement to become a routine parameter for diagnosis of inflammatory diseases.

Detection of native human complement components C3 and C5 and their primary activation peptides C3a and C5a (anaphylatoxic peptides) by ELISAs with monoclonal antibodies.

Monoclonal antibodies (mAbs) were raised against human C3a, C3b, C5a, and C5b after immunization of BALB/c mice with the native components C3 and C5. Using different combinations of these mAbs we have developed four sensitive sandwhich-enzyme-linked immunosorbent assays (ELISAs) for the detection of native C3 or C5 in samples with low concentrations of these proteins, e.g., in cell culture supernatants or synovial fluids and cerebrospinal fluids (CSF) and for the detection of the anaphylatoxic peptides (AT-peptides) C3a or C5a in human EDTA-plasma. The C3- and C5-ELISAs were found to be specific for the uncleaved complement proteins. Two different anti-C3a or anti-C5a mAbs were combined for the C3a- and C5a-ELISA. Before assaying a sample in the C3a- or C5a-ELISA a precipitation step to eliminate uncleaved C3 and C5 was necessary. The sensitivity and specificity of the four ELISAs were tested with purified antigens and EDTA-plasma or Cobra venom factor-activated EGTA-plasma samples as a source of C3a and C5a. The detection limits were 1 ng/ml for C3, 1 ng/ml for C3a, 2 ng/ml for C5, and 100 pg/ml for C5a. Plasma samples from patients undergoing cardiopulmonary bypass (CPB) surgery were used as a source of pathological material.

Rapid quantification of C3a and C5a using a combination of chromatographic and immunoassay procedures.

Monoclonal antibodies were isolated which reacted specifically with the complement cleavage products C3a, C3adR, C5a, and C5adR but not with the parent molecules C3 or C5. In both cases the mAbs showed a higher affinity towards the desArg forms. These mAbs were used as capture antibodies in immunoassays for C3a/C3adR and C5a/C5adR. The immunoassays are based on the ABICAP technology which ensures for a rapid measurement. Due to the large binding capacity and the very short diffusion pathways in the gel-matrix the binding equilibrium between capture antibodies and the antigen is reached whilst the sample is flowing through the column. Therefore this test represents an endpoint assay offering the possibility of using a single calibration curve for a large number of measurements. With the C3adR assay concentrations down to 16 ng/ml C3adR can be detected. The lower detection limit of the C5adR assay is 1 ng/ml C5adR. The tests for C3a/C3adR, and C5a/C5adR can be performed in 20 to 25 min and this rapid processing of plasma samples should permit the application of these parameters for diagnostic purposes and patient management.

Guinea pig C3 specific rabbit single chain Fv antibodies from bone marrow, spleen and blood derived phage libraries.

We constructed combinatorial immunoglobulin libraries from the whole rabbit antibody repertoire of bone marrow, spleen and peripheral blood of a rabbit immunized with guinea pig complement protein C3. By means of the phage display technology we selected guinea pig C3 specific single chain Fv (scFv) antibodies from each of the libraries. None of the scFv antibodies cross reacted with guinea pig C3a, human C3 or rat C3. The frequency of bone marrow derived C3 positive clones was much higher as compared to blood or spleen derived clones. Additionally bone marrow and spleen derived clones show higher diversity than clones, obtained from blood, as determined by fingerprint analysis with the restriction enzyme AluI. Dissociation rate constants for all scFvs were similar, indicating that the source of the scFvs had no influence on affinities. The antibody fragments were used to analyze complement activation during xenotransplantation. Several blood or bone marrow derived scFvs bound to C3 located on rat liver endothelium after hyperacute rejection of a heterotopically transplanted rat liver into guinea pig. These data demonstrate that monoclonal rabbit scFvs can be easily generated from recombinant phage display libraries, constructed from spleen, blood or bone marrow. The selected guinea pig C3 specific scFvs appear to be useful to detect complement activation during xenotransplantation in guinea pigs.

Tryptophan mutants of human C5a anaphylatoxin: a fluorescence anisotropy decay and energy transfer study.

Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys27 for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp2]rhC5a > [Trp64]rhC5a > [Trp70]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp70 to the 1,5-AEDANS group at Cys27 yielded a distance distribution of 2.4 +/- 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.

Chromatographic purification of human alpha 1 proteinase inhibitor from dissolved Cohn fraction IV-1 paste.

A novel chromatographic process for purification of alpha 1 proteinase inhibitor (alpha 1-PI) from Cohn fraction IV-1 paste is described. This process has been successfully scaled up to 50-1 columns. It involves DEAE chromatography, sulfopropyl (S) cation chromatography, tri-n-butyl phosphate (TNBP)-cholate treatment, a second S cation chromatography, freeze-drying and dry-heat. The process has been optimized for purity, yield, lipid removal, chemical usage and water consumption. Filtration after TNBP-cholate treatment plays a key role in ensuring a low lipid content in the final product. Pre-equilibration with high salt buffer is necessary to reduce the water consumption significantly during the ion-exchange chromatography equilibration step. The final product is approximately 95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a 64% to 70% yield from IV-1 paste.

[Energy expenditure in soldiers working on the public road and railroad construction]. / Wydatek energetyczny zolnierzy zatrudnionych przy budowie dróg publicznych, linii kolejowych oraz kolejowych pracach budowlanych.

The studies consisted in the determination of energetic expenditure of soldiers building roads and railway lines in different regions of the country and in different local conditions. In eight-hours' working days the calorific cost of 53 activities was determined. The studies involved 155 soldiers. The soldiers were occupied at earth work, surface work, production of asphalt and concrete, tract and sewage construction, operation of mechanized equipment. The energetic cost of the activities performed by the soldiers ranged between 3.1--13.0 kcal/min. and when converted per body weight kilogram 0.0435--0.1866 kcal/min/kg. As we miss the data on the energetic expenditure of workers building roads and railway lines the obtained results may be used at the ergonomic analysis of workplaces and at setting-up of an appropriate caloric value of food.

[Free forearm skin flaps with microvascular anastomosis for pharyngeal reconstruction]. / Wolny plat skóry przedramienia z mikrozespoleniem naczyn do rekonstrukcji gardla.

Free forearm skin flap with microvascular anastomosis was used for tissue defect reconstruction in 4 patients after resection of the oropharynx, base of the tongue and ramus of the mandible. The vascular pedicle of the flap contained the radial artery and the cephalic vein. For microvascular anastomosis the facial artery was used in all the patients, the facial vein in 2 patients, the internal jugular vein in one patient and the external jugular vein in the other one. Good healing of the graft was obtained in 3 patients. In one patient partial necrosis developed. No patient had fistula.

[Platysma myocutaneous flap in intraoral reconstruction]. / Plat miesnia szerokiego szyi w rekonstrukcji jamy ustnej.

Plastyma myocutaneous island flap was used in 13 patients to reconstruct tissue defect after oral cancer surgery. Necrosis of the skin island in the oral cavity occurred in 3 patients. A fistula developed in one patient with a large defect of the tongue, pharynx and mandible. In 12 patients wound healing on the neck was good. Plastyma myocutaneous flap is very useful to close defects of the floor of the mouth and up to the half of the tongue width.

[Operations of parotid malignant tumors]. / Operacje nowotworów zlosliwych slinianek przyusznych.

In a group of 64 patients with malignant tumours of the parotid gland the most common type was adenoid cystic carcinoma (20%) and malignant pleomorphic adenoma (14%). A group of 33 patients with follow-up from 4 months to 18 years was analyzed. The overall 5-year survival rate was 45%.

[Face deformation and function defects after maxillectomy]. / Znieksztalcenie twarzy i ubytki funkcji po maksylektomii.

In the group of 42 patients after maxillectomy face deformation and function defects were assessed. The best cosmetic and functional result was observed after maxillectomy without orbital exenteration, with preservation of lateral part of the zygomatic bone and at least a half of nasal skeleton. Severe face deformation developed after orbital exenteration, resection of the palpebrae, cheek and external nose.