Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis.

AIMS: To produce and characterize egg yolk immunoglobulin (IgY) against the fish intracellular pathogen Piscirickettsia salmonis as well as to evaluate the antibacterial activity of IgY in vitro and the availability in the serum of fish immunized orally. METHODS AND RESULTS: Specific IgY was produced by immunizing hens with P. salmonis proteins. The IgY was obtained from egg yolks using the ammonium sulphate precipitation method and it was characterized by SDS-PAGE, Western-blot and ELISA, demonstrating that anti-P. salmonis IgY strongly reacted specifically against P. salmonis proteins. In an in vitro neutralization assay, IgY inhibited the growth of P. salmonis in liquid medium at concentrations ranging from 128 to 256 µg ml(-1) in a dose-dependent manner. Interestingly, IgY against P. salmonis also generates a strong protective effect on the infection of P. salmonis in salmon head kidney-1 cells. In addition, the bacteriostatic function of IgY appears to result possibly from agglutination by the interaction of IgY with surface components of the pathogen. Finally, to confirm this IgY as an alternative for salmonid treatment, Atlantic salmon (Salmo salar) specimens were orally inoculated with IgY. The analysis of the sera demonstrates that IgY was effectively transported by fish intestine and that this immunoglobulins maintains its properties and recognizes several proteins of P. salmonis up to 12 h after inoculation of IgY against P. salmonis. CONCLUSIONS: Specific IgY effectively inhibited the growth of P. salmonis and this immunoglobulin can be released in the Atlantic salmon sera when administered orally to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose that this specific IgY against this fastidious micro-organism could be a useful strategy for the treatment of piscirickettsiosis.

Activation of kinin B1 receptors induces chemotaxis of human neutrophils.

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.

Fatal infection caused by Francisella tularensisin a neutropenic bone marrow transplant recipient.

Francisella tularensis is one of the most infectious pathogenic bacteria known. Even though immunity against this organism is thought to be primarily T cell mediated, some evidence suggests that neutrophils may also play an important protective role. We report a case of tularemia in a neutropenic bone marrow transplant recipient that sheds light on the importance of neutrophils in protection against this infection and review clinical aspects of this fascinating infection emphasizing areas of interest for immunocompromised hosts.

Formation of compound I and compound II ferryl species in the reaction of hemoglobin I from Lucina pectinata with hydrogen peroxide.

The formation of ferryl heme (Fe(IV) = O) species, i.e., compound I and compound II, has been identified as the main intermediates in heme protein peroxidative reactions. We report stopped-flow kinetic measurements which illustrate that the reaction of hemoglobin I (HbI) from Lucina pectinata with hydrogen peroxide produce ferryl intermediates compound I and compound II. Compound I appears relatively stable displaying an absorption at 648 nm. The rate constant value (k'(2)) for the conversion of compound I to compound II is 3.0 x 10(-2) s(-1), more than 100 times smaller than that reported for myoglobin. The rate constant value for the oxidation of the ferric heme (k'(12) + k'(13)) is 2.0 x 10(2) M(-1) s(-1). These values suggest an alternate route for the formation of compound II (by k'(13)) avoiding the step from compound I to compound II (k'(2)). In HbI from L. pectinata the stabilization of compound I is attribute to the unusual collection of amino acids residues (Q64, F29, F43, F68) in the heme pocket active site of the protein.

Effect of hypoxia and hypoxia/reoxygenation on proteoglycan metabolism by vascular smooth muscle cells.

Hypoxia and hypoxia/reoxygenation are known to affect vascular smooth muscle cell physiology. In this study, we first investigated proteoglycan synthesis by human aortic smooth muscle cells exposed to normoxia, hypoxia, or hypoxia/reoxygenation. We then compared the newly synthesized proteoglycans from normoxic and hypoxic-reoxygenation cultures for their ability to bind low density lipoprotein (LDL). Confluent smooth muscle cells under normoxia, hypoxia, or hypoxia/reoxygenation were pulsed with [35S]sulfate, and secreted and cell-associated proteoglycans were analyzed. Secreted proteoglycans in cultures exposed to hypoxia (4 h)/reoxygenation (19 h) increased 28% over those of cells continuously exposed to normoxia. Cell-associated proteoglycans did not differ significantly between the two groups. In contrast, hypoxia (4 h) followed by a 30-min reoxygenation produced a 37% decrease in newly synthesized proteoglycans. Hypoxia alone also resulted in a 24% decrease in secreted proteoglycans and a 20% decrease in cell-associated proteoglycans. Proteoglycans newly synthesized by smooth muscle cells exposed to normoxia and hypoxia/reoxygenation did not differ in their charge densities and molecular size but did differ in glycosaminoglycan composition. Exposure of smooth muscle cells to hypoxia/reoxygenation produced a 60% increase in a proteoglycan subfraction that bound LDL with very high affinity. The incorporation of [3H]leucine into total cellular protein decreased significantly following exposure of smooth muscle cells to hypoxia as well as hypoxia/reoxygenation. These results indicate that hypoxia and hypoxia/reoxygenation cause major alterations in proteoglycan metabolism by vascular smooth muscle cells.

Isolation, characterization, and distribution of a 24-kDa proteoglycan in the urine of cachectic cancer and AIDS patients.

Substantial weight loss in individuals with AIDS or cancer is associated with a poor prognosis and increased mortality. We have isolated and partially characterized a proteoglycan (named azaftig) from the urine of a cancer patient experiencing weight loss. Furthermore, we have raised a polyclonal antibody to azaftig in rabbits and developed a procedure to measure the level of this proteoglycan in urine by Western blot. We report the presence of azaftig in the urine of cancer and AIDS patients experiencing weight loss, but not in the control or weight-stable subjects. The azaftig-like immunoreactivity was present in 69.2% (9/13) of patients with weight loss, but only in 27.0% (3/11) of weight-stable cancer or AIDS patients and none of the control subjects (n = 8).

Altered composition and increased endothelial cell proliferative activity of proteoglycans isolated from breast carcinoma.

BACKGROUND AND OBJECTIVES: Proteoglycans of the extracellular matrix are vital to the growth and evolution of malignant neoplasms. The present study determined the composition of proteoglycans isolated from paired specimens of normal breast and adenocarcinoma of the breast harvested from each patient (n = 8). The proteoglycans were then tested for their ability to stimulate endothelial cell proliferation. METHODS: Proteoglycans were isolated by extraction with 4 M guanidine hydrochloride and purified by CsCl density-gradient centrifugation. The proteoglycans were characterized and tested for their ability to simulate endothelial cell proliferation. RESULTS: In each case, the total proteoglycan content of the tumor was significantly greater than that of the corresponding normal tissue. The proteoglycans isolated from the carcinoma contained 32.2% (13.7/42.5) more chondroitin sulfate, 18.5% (5.6/30.2) less dermatan sulfate, and 29.6% (8.1/27.3) less heparan sulfate than did the proteoglycans of normal breast tissue. Proteoglycans from normal tissue did not stimulate endothelial cell proliferation, whereas those from malignant tissue stimulated proliferation by 1.3- to 1.5-fold. CONCLUSIONS: These results indicate that malignant breast tissue exhibits both qualitative and quantitative changes in proteoglycan composition, which, in turn, may stimulate endothelial cell proliferation.

In vitro infection of smooth muscle cells by Chlamydia pneumoniae.

Recent observations have shown that both Chlamydia pneumoniae antigens and DNA may be found within atherosclerotic lesions. In this study, we evaluated the ability of C. pneumoniae to infect cells that make up atherosclerotic lesions, including endothelial cells, smooth muscle cells, and cholesterol-loaded smooth muscle cells. The organism readily infected rabbit, bovine, and human aortic smooth muscle cells. Cholesterol-loaded smooth muscle cells were even more susceptible to C. pneumoniae infection. Chlamydia trachomatis inefficiently infected smooth muscle cells, demonstrating that this is not a characteristic of all members of the genus Chlamydia. C. pneumoniae infected bovine endothelial cells poorly. This study demonstrates that C. pneumoniae readily infects one of the important types of cells found within atherosclerotic lesions, i.e., smooth muscle cells with and without cholesterol loading.

Enhanced synthesis of proteoglycans by vascular endothelial cells treated with phorbol ester.

We investigated the biosynthesis of proteoglycans (PG) in endothelial cells following their treatment with phorbol 12-myristate 13-acetate (PMA). Confluent cultures of bovine aortic endothelial cells were incubated in the presence and absence of PMA (100 ng/ml) and then pulsed with [35S]sulfate, [3H]glucosamine, or [35S]sulfate plus [3H]leucine for varying times in the absence of PMA. Alternatively, confluent endothelial cells were simultaneously incubated with PMA and [35S]sulfate for varying times. The metabolically labeled PG in the cell layer and medium were analyzed. Both short-term and prolonged exposure of endothelial cells to PMA significantly stimulated PG synthesis, regardless of the experimental conditions. [35S]sulfate incorporation into newly synthesized PG in PMA-treated cells also increased by 1.7-fold and 3.6-fold over control cells, following a 15-min and 30-min pulse, respectively. Cycloheximide markedly inhibited the increased synthesis of PG in PMA-treated cells, while actinomycin D produced a moderate inhibition. PG secretion was increased in PMA-treated cells compared with control cells, while there was no significant difference in PG degradation between the two cultures. PG from control and PMA-treated endothelial cell cultures did not differ in composition or hydrodynamic sizes. The incorporation of [3H]leucine into total cellular proteins decreased significantly following exposure of endothelial cells to PMA. Endothelial cells exposed to PMA for 3 h had significantly more protein kinase C (PKC) activity than did control cells. Inhibition of PKC by calphostin C abolished the PMA-mediated stimulation of PG synthesis in endothelial cells. The results indicate that PMA stimulates PG synthesis in endothelial cells either directly or indirectly through a PKC dependent mechanism.

Elevated expression of proteoglycans in proliferating vascular smooth muscle cells.

Smooth muscle cell (SMC) proliferation and increased production of arterial wall proteoglycans (PG) are implicated in atherogenesis. We investigated the effect of SMC proliferation on the biosynthesis of PG and the ability of the newly synthesized PG to bind low density lipoprotein (LDL). Proliferating and quiescent human aortic SMC were pulsed with [35S]sulfate for 24 h. Secreted and cell-associated PG were then analyzed. When SMC plated at a low density were induced to proliferate, PG synthesis increased significantly in comparison with quiescent cells. This was the net result of a 2.7-fold increase in secreted PG and a 1.3-fold increase in cell-associated PG. The increased PG synthesis in proliferating SMC correlated with a significant increase in the steady-state level of mRNA for perlecan and biglycan, and a modest increase in the versican-specific mRNA. The mRNA for decorin showed a 40% decrease. The increased PG secretion in proliferating cultures was due to increases in heparan sulfate PG, dermatan sulfate PG, and chondroitin sulfate PG secretion. Quiescent SMC at confluency produced 50% less PG than the corresponding SMC plated at a low density. Although confluent SMC stimulated to proliferate also had increased PG synthesis, this was 50% less than the PG synthesis by proliferating SMC that were initially plated at a low density. The PG synthesized by proliferating and quiescent SMC did not differ in charge density and molecular size. Secreted PG from both quiescent and proliferating cultures contained subfractions that bound LDL with high affinity. However, compared with quiescent cultures, the proliferating cultures produced more of a PG subfraction that exhibited very high affinity to LDL (31.6% in quiescent cultures versus 40.8% in proliferating cultures). These results indicate that PG metabolism is altered significantly in proliferating human SMC which might have implications in the pathophysiology of atherosclerosis.

Isolation and characterization of a proteoglycan variant from human aorta exhibiting a marked affinity for low density lipoprotein and demonstration of its enhanced expression in atherosclerotic plaques.

Proteoglycans (PG) are implicated in the pathophysiology of atherosclerosis due to their ability to complex with plasma low density lipoproteins (LDL). Studies were conducted to determine whether human aorta contains PG subclasses that exhibit enhanced LDL binding ability. PG were isolated from normal and atherosclerotic aortas by a combination of dissociative extraction and ion-exchange chromatography. The PG were further subfractionated on an LDL affinity column based on their binding affinity to LDL. Two PG fractions exhibiting high-affinity binding to LDL, as evidenced by their elution at 1.0 and 1.5 M NaCl, respectively, were isolated from both normal and atherosclerotic tissue. Compared with normal tissue, atherosclerotic tissue showed a twofold increase in the high-affinity PG that eluted at 1.5 M NaCl. Gel filtration of the high-affinity PG from normal tissue yielded two peaks (nPG2 and nPG3), while the high-affinity PG from plaque tissue was resolved into three peaks (pPG1, pPG2, and pPG3). pPG1 eluted at the void volume of the column, indicating that it was of very large molecular size. The hydrodynamic size of pPG2 was larger than that of the corresponding nPG2 (Kav = 0.44 versus 0.51), while pPG3 had the same hydrodynamic size as nPG3 (Kav = 0.86). The high-affinity PG subfractions from normal aorta contained varying proportions of chondroitin sulfates, dermatan sulfates, and heparan sulfate. In contrast, the PG subfractions from plaque tissue contained predominantly chondroitin sulfates and heparan sulfate. In vitro complexes of LDL and the high-affinity PG fractions from normal aorta and plaque tissue stimulated cholesteryl ester synthesis in human monocyte-derived macrophages. However, the LDL-plaque PG complex was significantly more potent than the LDL-normal aorta PG complex in this respect. These results indicate that PG subclasses with enhanced binding affinity to LDL occur in the normal human aorta and that their concentration increases significantly in atherosclerotic lesions. In addition, the high-affinity PG in plaque tissue have altered characteristics and increased ability to stimulate LDL-mediated cholesterol ester synthesis in macrophages. This could lead to increased lipid deposition during atherogenesis.

Marked alteration of proteoglycan metabolism in cholesterol-enriched human arterial smooth muscle cells.

To elucidate the correlation between vascular cholesterol metabolism and proteoglycan (PrGl) biosynthesis, we investigated PrGl synthesis in human aortic smooth muscle cells (SMCs) after cholesterol enrichment with cationized low-density lipoproteins (LDL). Compared with normal SMCs, total PrGl synthesis by cholesterol-enriched cells decreased 2.4-fold (11874 +/- 530 d.p.m. per 10(5) cells compared with 4890 +/- 385 d.p.m. per 10(5) cells). This was the net result of a 6.9-fold reduction in medium PrGl (11000 +/- 490 d.p.m. per 10(5) cells compared with 1580 +/- 246 d.p.m. per 10(5) cells) and a 3.8-fold increase in cellular PrGl over controls (874 +/- 27 d.p.m. per 10(5) cells compared with 3310 +/- 193 d.p.m. per 10(5) cells). Prior incubation of SMCs with native LDL had no effect on PrGl synthesis by these cells. The decrease in PrGl synthesis in cholesterol-enriched cells correlated with a 90% and 20% reduction in the steady-state level of mRNA for biglycan and decorin respectively, and a virtual elimination of the steady-state level of mRNA for versican over controls. Despite the down-regulation of PrGl synthesis, cholesterol-loaded cells produced a 2-fold increase in a PrGl subfraction with high affinity for LDL. Compared with the corresponding PrGl subfraction from normal cells, that from the cholesterol-enriched cells exhibited increased charge density and a higher molecular mass and contained relatively larger proportions of chondroitin 6-sulphate and dermatan sulphate. These results show that PrGl metabolism is dramatically altered in cholesterol-enriched human SMCs.

Invasive Haemophilus influenzae type b infection in a child with familial deficiency of the beta subunit of the eighth component of complement.

A child who had had meningitis caused by Haemophilus influenzae type b, and then had meningococcal meningitis, was found to have familial deficiency of the beta subunit of the eighth component of complement. The child had not received the H. influenzae type b vaccine. If this deficiency is discovered, we recommend that family members be screened, regardless of their health status.

Identification of a Renibacterium salmoninarum DNA fragment associated with bacterial internalization into CHSE-cultured cells.

We report here the isolation of a Renibacterium salmoninarum DNA sequence capable of transforming a non-invasive Escherichia coli strain into a microorganism able to enter the fish cell line, CHSE-214. Immunofluorescence and electron microscopy techniques were used to assess the acquired invasive phenotype by HB101 E. coli cells, upon transformation with pPMV-189. This plasmid carries a 2282-bp R. salmoninarum DNA segment. The invasive phenotype is conserved upon deletion of approximately 1000 bp at the 3' end of the insert. The remaining segment contains an ORF region encoding a putative protein of about 30 kDa.

Serratia marcescens peritonitis in a patient receiving chronic ambulatory peritoneal dialysis complicated by osteomyelitis.

Peritonitis, a common complication of chronic ambulatory peritoneal dialysis (CAPD), arises from various bacterial pathogens. We report the case of a patient with CAPD-associated peritonitis caused by Serratia marcescens. This case was further complicated by the development of osteomyelitis believed to have occurred from hematogenous seeding of Serratia marcescens.

Doppler echocardiographic assessment of valvular heart disease in patients requiring hemodialysis for end-stage renal disease.

Valvular heart disease accounts for considerable morbidity and some mortality among patients with end-stage renal disease (ESRD). To determine the prevalence and severity of valvular and perivalvular abnormalities, including valvular thickening, stenosis, calcification, regurgitation, and mitral annular calcification (MAC), and the impact of age and duration of dialysis on valvular disease in these patients, we reviewed Doppler echocardiograms in 75 consecutive patients requiring hemodialysis for ESRD. Our results showed that valvular sclerosis, calcification, and MAC were common in patients requiring hemodialysis; valvular regurgitation was both more prevalent and more severe in hemodialysis patients than in "normal subjects"; and age of the patient and duration of dialysis were related to the prevalence of valvular disease in ESRD. These findings suggest that Doppler echocardiography should be frequently used to assess possible valvular heart disease in patients receiving hemodialysis for ESRD, particularly older patients, those who have received dialysis for several years, and those with suspected valvular disease based on history and physical examinations.

Infectious diseases associated with complement deficiencies.

The complement system consists of both plasma and membrane proteins. The former influence the inflammatory response, immune modulation, and host defense. The latter are complement receptors, which mediate the cellular effects of complement activation, and regulatory proteins, which protect host cells from complement-mediated injury. Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components. Four aspects of the complement cascade are critical to its function and regulation: (i) activation of the classical pathway, (ii) activation of the alternative pathway, (iii) C3 convertase formation and C3 deposition, and (iv) membrane attack complex assembly and insertion. In general, mechanisms evolved by pathogenic microbes to resist the effects of complement are targeted to these four steps. Because individual complement proteins subserve unique functional activities and are activated in a sequential manner, complement deficiency states are associated with predictable defects in complement-dependent functions. These deficiency states can be grouped by which of the above four mechanisms they disrupt. They are distinguished by unique epidemiologic, clinical, and microbiologic features and are most prevalent in patients with certain rheumatologic and infectious diseases. Ethnic background and the incidence of infection are important cofactors determining this prevalence. Although complement undoubtedly plays a role in host defense against many microbial pathogens, it appears most important in protection against encapsulated bacteria, especially Neisseria meningitidis but also Streptococcus pneumoniae, Haemophilus influenzae, and, to a lesser extent, Neisseria gonorrhoeae. The availability of effective polysaccharide vaccines and antibiotics provides an immunologic and chemotherapeutic rationale for preventing and treating infection in patients with these deficiencies.

Membranous nephropathy in rheumatoid arthritis.

We have described a man with classic rheumatoid arthritis who had membranous nephropathy associated with nephrotic syndrome not related to gold or penicillamine therapy. Complete remission of the nephrosis occurred after discontinuation of hydroxychloroquine therapy. Further study is needed to determine the incidence of subclinical membranous nephropathy in patients with rheumatoid arthritis, and the effect of drugs in triggering clinical manifestations of this condition.