Effect of recombinant basic fibroblast growth factor (bFGF) on fibroblast-like cells from human rotator cuff tendon

Rotator cuff tendon cells (RCC) derived from surgical samples showed fibroblast-like morphology. Histological staining demonstrated collagen secretion by RCC. Immunohistological findings revealed that RCC secreted type I and III collagen, but not type II collagen. In addition, the SDS-PAGE analysis suggested that RCC predominantly produced type I collagen. Basic fibroblast growth factor (bFGF) had a stimulatory effect on the proliferation of RCC dose-dependently up to 1 ng/ml. Administration of bFGF suppressed the secretion of collagens from RCC in a dose-dependent manner.     

Structural heterogeneity of the C3b/C4b receptor (Cr 1) on human peripheral blood cells

In these studies CR 1 polymorphism previously demonstrated on erythrocytes (E) was also found on CR 1-bearing peripheral blood leukocytes including polymorphonuclear (PMN), eosinophils, monocytes, and B lymphocytes. However several cell-specific differences in CR1 were found: (a) an approximately 5,000-dalton increase in CR 1 on PMN and eosinophils, (b) unequal band intensity among heterozygotes suggests that there is preferential expression of 220,000- or 225,000-dalton receptors on leukocytes compared to E, and (c) "minor" bands, approximately 15,000 daltons larger than the major receptor molecule, were found on E but not on leukocytes. These observations constitute a unique example of heterogeneity of an integral membrane receptor.     

High-molecular-weight hydroxyproline-protein in relation to immunoglobulins M (IgM) in human blood serum.

The content of the high-molecular-weight protein-bound hydroxyproline (Hyp) and IgM level in the sera of 30 healthy persons and 29 patients showing changes in gamma and/or beta globulin levels was determined. Statistically significant correlations between quantities of these components have been found. On the base of the present results as well as the results of previous studies authors suggest that the high-molecular-weight Hyp-protein contains immunoglobulin of the IgM class.     

3-Mercaptopyruvate sulfurtransferase (EC 2.8.1.2): a simple assay adapted to human blood cells

A product of cysteine catabolism, 3-mercaptopyruvate, is enzymatically degraded to sulfur and pyruvate by a sulfurtransferase (EC 2.8.1.2.) present in human red and white blood cells. A simple sulfurtransferase assay is reported which takes advantage of the fact that pyruvate is conveniently measured enzymatically by lactate dehydrogenase (LDH) after the elimination of 3-mercaptopyruvate, also a substrate of LDH, by addition of N-ethylmaleimide. Sulfite is employed as sulfur acceptor, and conditions for a reproducible assay including data for preparation and storage of reactants, their assay, and their optimal concentrations are given. The apparent K_m for sulfite is 6.5 · 10^?3 M and for 3-mercaptopyruvate is 1.9 · 10^?3 M. A reducing agent, in this assay dithiothreitol (Cleland's reagent), is essential for active transsulfuration. A normal metabolite, 3-mercaptopyruvate is reported elsewhere to have the capacity of producing polyploidy and chromosomal endoreduplication, a feature rendering it of interest in tumor metabolism.     

Interference of the measurement of lactate dehydrogenase (LDH) activity in human serum and plasma by LDH from blood cells

This study was performed to find a reliable method to measure lactate dehydrogenase (LDH) activity in blood samples. Human plasma contains thrombocytes whose number depends on the speed of centrifugation applied to the freshly-drawn blood sample. They do not disturb the measurement of LDH in plasma, provided the plasma sample has not been subjected to procedures that may destroy the integrity of the thrombocytes, e.g. standing overnight at 4 degrees C, freezing, or treatment with ultrasonic waves. Centrifugation of plasma at a minimum of 1174 x g eliminates the presence of thrombocytes. The serum of healthy human subjects invariably contains higher LDH activity than the plasma: the difference is 30 +/- 18 U/l (mean +/- S.D.). LDH-isoenzyme analysis shows that a large part of the LDH activity difference is caused by lysis of erythrocytes in the clot. Liberation of 30 U LDH per litre is not associated with visible hemolysis.     

Cytotoxic and antioxidative potentials of ethanolic extract of Eugenia uniflora L. (Myrtaceae) leaves on human blood cells

Eugenia uniflora is used in the Brazilian folk medicine to treat intestinal disorders and hypertension. However, scanty information exist on its potential toxicity to human, and little is known on its antioxidant activity in biological system. Hence, we investigated for the first time the potential toxic effects of ethanolic extract (EtOH) of E. uniflora (EEEU) in human leukocytes and erythrocytes, as well as its influence on membrane erythrocytes osmotic fragility. In addition, EEEU was chemically characterized and its antioxidant capacity was evaluated. We found that EEEU (1–480 μg/mL) caused neither cytotoxicity nor DNA damage evaluated by Trypan blue and Comet assay, respectively. EEEU (1–480 μg/mL) did not have any effect on membrane erythrocytes fragility. In addition, EEEU inhibited Fe²⁺-induced lipid peroxidation in rat brain and liver homogenates, and scavenged the DPPH radical. EEEU presented some polyphenolic compounds with high content such as quercetin, quercitrin, isoquercitrin, luteolin and ellagic acid, which may be at least in part responsible for its beneficial effects. Our results suggest that consumption of EEEU at relatively higher concentrations may not result in toxicity. However, further in vitro and in vivo studies should be conducted to ascertain its safety.     

Hemagglutination inhibition studies for the evaluation of blood group antigens in ethanol soluble substances (ESS) obtained from human, baboon and vervet monkey red blood cells

Soluble blood group substances, isolated from the red blood cells of humans, baboons, and vervet monkeys by ethanol extraction, possessed serologically active specificities for the following antigens: A, B, H, Lea, LebL, P, P19 Pk and I. Human red blood cells lacking any of these specificities by the direct hemagglutination test also lacked the related antigens in their soluble extract. The only exception was in "Bombay" Oh cells, from which soluble H substance could be readily isolated. Soluble substances obtained from baboon and vervet monkey red blood cells, which lack the human variety of A, B, and H antigens on their red blood cells, inhibited both human and lectin anti-H reagents. The detection of "hidden" H activity in Oh cells will pose some important questions regarding membrane characteristics and the role of immune surveilance.     

Immunologic responsiveness of the C3H/HeJ mouse: differential ability of butanol-extracted lipopolysaccharide (LPS) to evoke LPS-mediated effects

The lipopolysaccharide (LPS)-protein complex extracted from the cell wall of Escherichia coli K235 by the butanol-water technique has been shown to evoke a mitogenic response in bone marrow-derived (B) lymphocytes from the C3H/HeJ mouse strain. These mice are resistant to the effects of LPS extracted with phenol. Therefore, the ability of butanol-extracted LPS to modulate a spectrum of C3H/HeJ B-cell functions was investigated. Both butanol-extracted (LPS-B) and phenol-extracted (LPS-P) LPS preparations activated responder C3H/St spleen cell cultures to polyclonal antibody production, while only LPS-B activated C3H/HeJ spleen cells. Both LPS-P and LPS-B acted as adjuvants when injected after aggregated human gamma globulin (HGG) in C3H/St mice, but neither preparation was effective as a adjuvant in C3H/HeJ mice. LPS-P injected with deaggregated HGG (tolerogen) into LPS-sensitive mice has been shown previously to inhibit the induction of tolerance HGG. In the present studies, it was shown that LPS-B, but not LPs-p, was able to inhibit tolerance induction to HGG in the C3H/HeJ, whereas both preparations were effective in the C3H/St. LPS has also been shown to bypass tolerant T cells in LPS-sensitive mice late in tolerance to HGG at a time when B cells are responsive. However, in the C3H/HeJ, neither LPS-B nor LPS-P was capable of this function. The responsiveness of these B cells to HGG was demonstrated in transfer experiments. Thus, in the C3H/HeJ, LPS-B stimulates mitogenesis, polyclonal B-cell activation, and inhibition of tolerance induction, but cannot act as an effective adjuvant or as a bypass mechanism to activate B cells in the presence of tolerant T cells. The explanation for this pattern of responses may be attributable to yet another cellular defect in the C3H/HeJ mouse.     

Binding of fluid phase C3b to nonsensitized bystander human red cells. A model for in vivo effects of complement activation on blood cells

There is evidence that activated complement fragments (C3b) can bind to human red cells (RBCs) serving as adsorbing surfaces, but not as antigens. This evidence prompted the present in vitro experiments. Using the standard antiglobulin test (AGT), the radioimmune antiglobulin test (RIAT), and the immune adherence test, we found that C3 can indeed be attached to "bystander" human RBCs if complement is activated either through the classical pathway (anti-A hemolysins plus blood group A1 RBCs) or the alternative pathway (activator surfaces, i.e., Escherichia coli bacteria, rabbit RBCs, or inulin). On Scatchard plot analysis, between 24,000 and 44,000 radiolabeled anti-C3d molecules were bound per one adjacent "bystander" RBC, while untreated control RBCs, or RBCs preincubated with fresh compatible serum, bound only 200 to 300, and 600 to 800 molecules, respectively. Despite strong coating with C3 fragments, only "bystander" paroxysmal nocturnal hemoglobinuria, but not normal, RBCs were hemolyzed by complement activation, i.e., through E. coli at pH 8.0. RBCs were not coated with C3 when complement was activated in the fluid phase by heat-aggregated IgG or a staphylococcal "decomplementation antigen." We conclude that the findings of our in vitro experiments accurately mimic some old, but as yet insufficiently understood, in vivo effects of complement activation on circulating blood cells.     

吉林省2018-2020年甲型H3N2亚型流感病毒耐药基因分析

目的 掌握2018-2020年吉林省内H3N2亚型流感流行株对金刚烷胺类药物以及神经氨酸酶抑制剂类药物的耐药情况,为流感的临床治疗及预防控制提供参考依据.方法 选取甲型H3N2亚型流感毒株34株,对M2和NA基因进行PCR扩增,测序后对耐药位点进行分析.结果 2018-2020年吉林省分离到流感毒株981株,其中甲型流...     

Pathogenesis of Campylobacter fetus infections. Failure of encapsulated Campylobacter fetus to bind C3b explains serum and phagocytosis resistance.

Campylobacter fetus ssp. fetus strains causing systemic infections in humans are highly resistant to normal and immune serum, which is due to the presence of high molecular weight (100,000, 127,000, or 149,000) surface (S-layer) proteins. Using serum-resistant parental strains (82-40 LP and 23D) containing the 100,000-mol wt protein and serum-sensitive mutants (82-40 HP and 23B) differing only in that they lack the 100,000-mol wt protein capsule, we examined complement binding and activation, and opsono-phagocytosis by polymorphonuclear leukocytes. C3 consumption was similar for all four strains but C3 was not efficiently bound to 82-40 LP or 23D even in the presence of immune serum, and the small amount of C3 bound was predominently the hemolytically inactive iC3b fragment. Consumption and binding of C5 and C9 was significantly greater for the unencapsulated than the encapsulated strains. Opsonization of 82-40 HP with heat-inactivated normal human serum caused greater than 99% killing by human PMN. Similar opsonization of 82-40 LP showed no kill, but use of immune serum restored killing. Findings in a PMN chemiluminescence assay showed parallel results. Association of 32P-labeled 82-40 HP with PMN in the presence of HINHS was 19-fold that for the 82-40 LP, and electron microscopy illustrated that the difference was in uptake rather than in binding. These results indicate that presence of the 100,000-mol wt protein capsule on the surface of C. fetus leads to impaired C3b binding, thus explaining serum resistance and defective opsonization in NHS, mechanisms that explain the capacity of this enteric organism to cause systemic infections.     

Development of a DNA probe from the deoxyribonucleotide sequence of a 3-N-aminoglycoside acetyltransferase [AAC(3)-I] resistance gene.

The aacC1 gene encoding the 3-N-aminoglycoside acetyltransferase [AAC(3)-I] was cloned from enteric plasmid pJR88, and its deoxyribonucleotide sequence was determined. Significant nucleotide homology was noted in the region extending from the proposed -35 sequences through the first 59 base pairs of the aacC1 gene open reading frame (ORF) and the upstream flanking regions and ORFs of several other antibiotic resistance genes. Sequences were noted to be homologous with the 6'-N-aminoglycoside acetyltransferase [AAC(6')-I], 2'-O-aminoglycoside adenylyltransferase [AAD(2')], and 3'-O-aminoglycoside adenylyltransferase [AAD(3')] resistance genes; the OXA-1, OXA-2, and PSE-2 beta-lactamase genes; and several dihydrofolate reductase genes. Small regions of homology were noted in the 3'-flanking regions of these resistance genes as well. A DNA probe for the aacC1 gene was selected from the nucleotide sequence information and was tested against a series of genetically and enzymatically defined strains. The probe, which proved specific for the aacC1 gene, was then tested against a series of 58 gentamicin-susceptible and 219 gentamicin-resistant gram-negative bacilli isolated from patients at the Seattle Veterans Administration Medical Center. Only six clinical isolates were noted to carry the aacC1 gene. Each was resistant to gentamicin but susceptible to kanamycin, tobramycin, and amikacin. The presence of homologous regions of DNA at both the 3' and 5' ends of the aacC1 gene reinforces the importance of choosing probes from within the ORFs of genes and of avoiding flanking sequences. When the homology with other sequences extends into the ORF, as it does with the aacC1 gene, development of a specific probe may require determination of the nucleotide sequence.     

Interactions of the third component of complement (C3) with cross-linked dextran. II. Demonstration of an alternate pathway activation as binding mechanism of C3 to cross-linked dextran.

In previous investigations we could show that incubation of cross-linked dextran (Sephadex) with normal human serum results in the binding of the third component of complement to the Sephadex beads. In this paper, data are presented which demonstrate that not only human but also guinea pig C3 reacts with Sephadex and that this binding is due to an alternate pathway of C3 activation. This conclusion was drawn, since a) C3 is bound also from guinea pig serum with total deficiency of C4, b) the reaction can be completely blocked by EDTA but only diminished by EGTA and c) the reaction turned out to be temperature dependent with an optimum at 37 degrees C and could be abolished by diluting the serum more than 1:16. The ability of cross-linked dextran to activate C3 via the alternate pathway seems to be due to conformational changes, since in our experiments soluble dextran of the same source was found to be ineffective in this respect. Implications from these findings and possible applications are discussed.     

Assessment of insulin action in insulin-dependent diabetes mellitus using [6(14)C]glucose, [3(3)H]glucose, and [2(3)H]glucose. Differences in the apparent pattern of insulin resistance depending on the isotope used.

To determine whether [2(3)H], [3(3)H], and [6(14)C]glucose provide an equivalent assessment of glucose turnover in insulin-dependent diabetes mellitus (IDDM) and nondiabetic man, glucose utilization rates were measured using a simultaneous infusion of these isotopes before and during hyperinsulinemic euglycemic clamps. In the nondiabetic subjects, glucose turnover rates determined with [6(14)C]glucose during insulin infusion were lower (P less than 0.02) than those determined with [2(3)H]glucose and higher (P less than 0.01) than those determined with [3(3)H]glucose. In IDDM, glucose turnover rates measured with [6(14)C]glucose during insulin infusion were lower (P less than 0.05) than those determined with [2(3)H]glucose, but were not different from those determined with [3(3)H]glucose. All three isotopes indicated the presence of insulin resistance. However, using [3(3)H]glucose led to the erroneous conclusion that glucose utilization was not significantly decreased at high insulin concentrations in the diabetic patients. [6(14)C] and [3(3)H]glucose but not [2(3)H]glucose indicated impairment in insulin-induced suppression of glucose production. These results indicate that tritiated isotopes do not necessarily equally reflect the pattern of glucose metabolism in diabetic and nondiabetic man.     

Immunologic properties of bacterial lipopolysaccharide (LPS). II. The unresponsiveness of C3H/HeJ Mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS

The C3H/HeJ mouse strain, previously shown to be a nonresponder to bacterial lipopolysaccharide (LPS)-induced mitogenesis in vitro, was demonstrated by the present studies to be competent to respond mitogenically to LPS, but only to LPS preparations obtained by selected extraction methods. These preparations appear to be confined to LPS isolated by mild extraction techniques, such as TCA or butanol. In contrast, those obtained by techniques utilizing phenol were only weakly stimulatory or completely nonstimulatory for spleen cells from the C3H/HeJ. All LPS preparations tested, on the other hand, were highly stimulatory for cells from another mouse strain, namely the C3H/St. The critical importance of the method of extraction of LPS on its mitogenic activity for C3H/HeJ cells was stressed by experiments in which LPS was prepared from Escherichia coli K235 using either of two procedures. In these experiments, phenol-extracted LPS, although mitogenic in the C3H/St, was completely nonstimulatory in the C3H/HeJ; whereas, butanol-extracted LPS was highly stimulatory in both strains of mice. This striking difference was attributed to a destructive effect of phenol on LPS, as demonstrated by the fact that treatment of butanol LPS with phenol resulted in a total loss of its mitogenic activity in the C3H/HeJ, but in only a partial loss in the C3H/St. In general, the mitogenic response observed with selected LPS preparations in the C3H/HeJ was quantitatively lower and more transient than that seen with the C3H/St, although qualitatively these responses appeared to be similar. This was evidenced by the observation that in both mouse strains LPS was a specific mitogen for B cells, a property which was also attributed in both strains to the same distinct structural region of the LPS molecule, that is lipid A. A preparation of LPS that failed to stimulate B cells from the C3H/HeJ nonetheless had the capacity to block activation of these B cells by a stimulatory preparation of LPS. These results strongly suggest that mitogenic stimulation of B cells by LPS is a function of the structural integrity of both the LPS molecule and putative B-cell receptors for LPS.     

Effect of human beta (s)-globin chains on cellular properties of red cells from beta-thalassemic mice.

The transgenic mouse system provides an in vivo setting in which to examine the effects on mouse red cells of hemoglobin genes that have been genetically introduced into the animals' genome. In this report, we have analyzed the cellular properties of red cells from homozygous beta-thalassemic mice (Hbbth-1/Hbbth-1), homozygous beta-thalassemic transgenic mice containing a human beta-sickle (beta(s)) gene (Hbb(th-1)/Hbb(th-1) + beta(s)), and normal animals. The presence of human beta(s)-globin chains in red cells from the Hbbth-1/Hbb(th-1) + beta(s) transgenic animals was noted to have a significant effect on cellular deformability and density distribution, as well as on the degree of anemia in these animals. We conclude from these studies that red cell deformability and density distribution is a sensitive means for assessing at the cellular level the effects of globin genes genetically introduced into whole organisms. In addition, these studies suggest that small decreases in the amount of excess alpha-globin chains can significantly ameliorate the severity of anemia in the beta-thalassemic mouse.