bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunological characterization of Vibrio cholerae O:1 lipopolysaccharide, O-side chain, and core with monoclonal antibodies
Authors: Gustafsson B , Holme T
Published: January 2020

Lipopolysaccharide (LPS) was extracted from Vibrio cholerae O:1 strains of the serotypes Ogawa, Inaba, and Hikojima and delipidated by mild-acid hydrolysis. Two polysaccharide fragments with the molecular weights of approximately 9,000 and 900, respectively, were isolated by gel permeation chromatography. The LPS preparations and the polysaccharide fragments were studied in enzyme-linked immunosorbent assay inhibition, rocket immunoelectrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroblotting with monoclonal antibodies directed against the group-specific antigen A, the type-specific antigens B (Ogawa) and C (Inaba), and the core region. Antigen A was demonstrated in all LPS preparations and all 9,000-molecular-weight fragments tested. The type-specific antigens B and C were demonstrated in LPSs and 9,000-molecular-weight fragments from Ogawa and Inaba, respectively. Furthermore, antigens B and C were both demonstrated in LPSs and 9,000-molecular-weight fragments from two of four Hikojima strains tested. Core antigen was demonstrated in the LPS and in the 9,000- and 900-molecular-weight fragments. The results indicate that the 9,000-molecular-weight fragment represents the complete polysaccharide chain, including group- and type-specific antigens as well as core antigens, whereas the 900-molecular-weight fragment constitutes the main part of the core region.
Prevention of urinary tract infection in rats with an indigenous Lactobacillus casei strain
Authors: Reid G , Chan R C , Bruce A W , Costerton J W
Published: January 2020

Our previous studies have shown that indigenous bacteria are able to block the in vitro attachment of uropathogenic bacteria to human uroepithelial cells. In the present study, we applied the concept of competitive exclusion to an animal model. A chronic urinary tract infection was established in female rats with bacteria incorporated into agar beads injected periurethrally into the urinary bladder via a no. 3 French ureteral catheter. Five strains of uropathogenic organisms were used in the first set of experiments, and their colonization of the bladder and kidneys of the animals was confirmed up to 2 months after injection. The uropathogens stimulated an immune response, detected by serum antibodies against the uropathogens, and an inflammatory response noted in sections of the kidneys stained with hematoxylin and eosin. Using this animal model, we established the persistent adherence of bacteria in the urinary tract without the need for creation of obstruction or implantation of a foreign body. In a second set of experiments, an isolate of Lactobacillus casei GR1 taken from the urethra of a healthy woman was incorporated into agar beads, instilled within the rat bladders on day 1, and then swabbed twice weekly for 21 days onto the introitus before challenge with uropathogens instilled into the urinary bladder. In 21 of 25 animals, no uropathogenic bacteria were recovered from the bladder and kidney tissues up to 60 days after challenge, and no immune response was detected. Our results show that L. casei prevented onset of urinary tract infection in 84% of the animals tested. The lactobacilli appeared to exclude the uropathogens from colonizing the urinary tract, within the first 48 hours after challenge, and the net effect was a complete eradication of bacteria from the uroepithelium. It is hoped that the demonstration of a protective role for indigenous bacteria in preventing urinary tract infection in an animal model will lead to the application of this technology to prevent recurrent urinary tract infection in female patients.
Regulation of egg antigen-induced in vitro proliferative response by splenic suppressor T cells in murine Schistosoma japonicum infection
Authors: Stavitsky A B , Olds G R , Peterson L B
Published: January 2020

Beginning about 5 weeks after infection, C57BL/6J mice infected with Schistosoma japonicum developed granulomas around parasite eggs trapped in the liver. These granulomas attained peak size about 9 weeks after infection and then spontaneously regressed. This regression was also induced by the injection of serum immunoglobulin G1 but not lymphoid cells from chronically infected mice, but it was conceivable that lymphoid cells from mice infected for 10 weeks could also induce regression. We investigated the possibility of cellular suppression of egg antigen-induced immune responses by coculturing spleen cells from 5- to 6-week-infected mice with spleen cells from mice infected for 10 weeks or longer. Mitomycin C-resistant Thy 1.2+, Lyt 2.2+ splenic T cells from mice infected for 10 to 25 weeks consistently suppressed the egg antigen-stimulated proliferation of spleen cells from 5- to 6-week-infected mice. Suppression was dependent upon specific antigen and optimal concentrations of egg antigen and T suppressor cells. Once induced, the suppressor cells were nonspecific. Cultured T cells from uninfected mice also occasionally suppressed the acute spleen cell proliferative response, but these cells were mitomycin C sensitive. These in vitro observations suggest that granulomatous inflammation in vivo may also be down regulated by suppressor T cells and that these cells may also be implicated in the nonspecific depression of cellular and humoral responses to antigens observed during the course of this infection.
Isolation of Staphylococcus aureus clumping factor
Authors: Espersen F , Clemmensen I , Barkholt V
Published: January 2020

Immunochemically identical components were isolated from water-soluble phases of five Staphylococcus aureus strains by affinity chromatography on fibrinogen-linked Sepharose 4B. The elution was performed with 1 M MgCl2. The component could be isolated from sonicated preparations of whole cells, cell walls, and extracellular products of S. aureus but not from sonicated preparations of staphylococcal L-forms or from Staphylococcus epidermidis. Investigations of the eluted component by immunoelectrophoresis and Western blot analysis by use of different polyspecific antibodies to S. aureus raised in rabbits revealed only one immunoprecipitate or one band. By means of gel filtration on Sepharose CL 6B and sodium dodecyl sulfate-polyacrylamide gel electrophoresis a molecular mass of 420,000 and 360,000 was found, respectively. Chemical analysis showed a carbohydrate content of about 20% by weight. By crossed immunoelectrophoresis the isolated component was demonstrated to bind to human fibrinogen. The finding that this purified component inhibited the fibrinogen-induced clumping of staphylococci strongly suggests that the component is the S. aureus clumping factor.
Kinetics and characterization of interferon production by murine spleen cells stimulated with Legionella pneumophila antigens
Authors: Blanchard D K , Klein T W , Friedman H , Stewart W E
Published: January 2020

Formalin-killed Legionella pneumophila bacterial cells, as well as a purified cell wall preparation (designated F-1 antigen) containing lipopolysaccharide (LPS), stimulated production of interferons (IFNs) in mouse spleen cell cultures. L. pneumophila whole-cell vaccine induced an IFN that was pH 2 labile and neutralized by anti-IFN-gamma indicating that IFN-gamma was the dominant form present. F-1 antigen induced a mixture of IFNs, depending upon the age of the culture and cell types present. In freshly prepared whole-spleen cultures and in 2-h adherent cultures, F-1 induced predominantly IFN-alpha/beta. In whole-spleen cultures that were allowed to age for 24 to 48 h before stimulation, F-1 was seen to induce mostly IFN-gamma, with low levels of IFN-alpha/beta present. Since only IFN-alpha/beta was produced in T-cell-depleted populations (at 2 h or at 48 h), it is suggested that T cells are responsible for IFN-gamma production in aged cultures. Additionally, heat-treated F-1, Escherichia coli LPS, and heat-treated E. coli LPS all induced similar levels of IFN-gamma in whole-splenocyte or nonadherent cell cultures which were incubated 48 h before stimulation. This suggests that LPS present in F-1 is responsible for IFN-gamma production and that an activated cell population is required. These results show that L. pneumophila antigens can induce the production of various types of IFN in mouse spleen cell cultures through several mechanisms.
Functional heterogeneity of intestinal Escherichia coli strains expressing type 1 somatic pili (fimbriae): assessment of bacterial adherence to intestinal membranes and surface hydrophobicity
Authors: Sherman P M , Houston W L , Boedeker E C
Published: January 2020

Although the role of host-specific, nonmannose-sensitive pilus adhesins in the intestinal adherence of pathogenic Escherichia coli is well established, a similar role for mannose-sensitive type 1 or common pili is less clear, since these structures can be expressed by most E. coli, even nonpathogens. We first examined whether type 1 pili, expressed by the rabbit-effacing, adherent, enteropathogenic E. coli strain RDEC-1, mediated interactions with intestinal membranes of several species and compared these interactions with those mediated by the nonmannose-sensitive adhesin of RDEC-1. We next grew a series of E. coli intestinal strains in static broth to promote type 1 pilus expression and determined whether E. coli expressing type 1 pili differed in their affinity for intestinal membranes (as measured by phase-contrast microscopy and aggregometry), hydrophobic surface properties, net negative surface charge (as measured by hydrophobic interaction chromatography and salt aggregation), and hemagglutination patterns. In contrast to the species-specific attachment to rabbit brush borders of RDEC-1 expressing its nonmannose-sensitive adhesin, type 1 pili on RDEC-1 mediated mannose-sensitive attachment to intestinal membranes of all four species tested. Expression of type 1 pili on other E. coli strains resulted in varying degrees of nonspecies-specific, mannose-sensitive attachment to intestinal membranes. This attachment correlated with increasing surface hydrophobicity rather than with hemagglutination patterns. These results indicate that various E. coli strains expressing type 1 pili are functionally heterogeneous and suggest that some type 1 pili might contribute to in vivo enteroadherence.
An in vitro ultrastructural study of infectious kidney stone genesis
Authors: McLean R J , Nickel J C , Noakes V C , Costerton J W
Published: January 2020

A ureolytic strain of Proteus mirabilis, isolated from a patient with infectious kidney stones, produced struvite (MgNH4PO4 X 6 H2O) and apatite [Ca10(PO4)6CO3] crystals in vitro when grown in artificial urine. Surface-attached crystals were encased in a slime-like layer. Scanning electron microscopy revealed that surfaces submerged in the artificial urine were colonized by P. mirabilis. Bacteria-associated crystals appeared soon after colonization and eventually became coated with an amorphous substance. Energy-dispersive X-ray analysis of these crystals revealed the presence of Mg, Ca, and P which are major components of struvite and apatite. Transmission electron microscopy of surface scrapings revealed that the glycocalyx of P. mirabilis contained a large number of crystals. Based on these observations and previous work, a theory for infectious renal calculogenesis is proposed. The kidney is initially colonized by invading ureolytic pathogens. These pathogens secrete copious amounts of glycocalyx which facilitates adhesion of the organisms to the kidney, provides protection for these bacteria, and serves to bind struvite and apatite crystals that result from bacterial urease activity. Growth of these calcified microcolonies into mature stones is characterized by continued bacterial growth, incorporation of urinary mucoproteins into the matrix along with bacterial glycocalyx, and a continued deposition of struvite and apatite crystals due to the high pH. The mature stone, in effect, represents an enlarged "fossilized" bacterial microcolony.
Measurement of streptococcal cell wall in tissues of rats resistant or susceptible to cell wall-induced chronic erosive arthritis
Authors: Anderle S K , Allen J B , Wilder R L , Eisenberg R A , Cromartie W J , Schwab J H
Published: January 2020

The quantity of streptococcal cell wall localized in the joints of rats of strains which are either susceptible (Sprague-Dawley, LEW/N, M520/N) or resistant (Buffalo, WKY/N, F344/N) to cell wall-induced chronic erosive arthritis was measured after intraperitoneal injection of group A streptococcal cell wall fragments. Susceptibility or resistance was not associated with a difference in the amount of cell wall localized in limbs or other tissues. It is concluded that although localization of cell wall in joint tissue is essential for development of arthritis, the relative resistance of certain rat strains reflects genetic regulation of inflammatory response rather than a quantitative difference in localization of cell wall in joints.
Antiglobulins in Endocarditis
Authors: Phair John P. , Klippel Jack , Mackenzie Malcolm R.
Published: January 2020

Twenty-five patients with endocarditis and serum antiglobulin activity were studied. The antiglobulins were detected by use of the latex agglutination reaction; only eight sera reacted with sensitized sheep cells and one with human O CD erythrocytes coated with anti-CD (Ripley) antibody. Absorption of the sera with the infecting organisms produced a decrease in antiglobulin activity in 10 of these sera. Absorption with other reference bacteria did not lower the titer of latex agglutination in 9 of 10 of these sera. No inhibition of antiglobulin activity by autologous immunoglobulin G was demonstrable. These findings suggest that some antiglobulins interact with the immunoglobulin G-bacterial complex and are analogous to anti-antibodies.
Foot-and-Mouth Disease Virus: Selection by Homogenized Calf Kidney Adsorption and Cell Culture Passage
Authors: Campbell C. H.
Published: January 2020

Foot-and-mouth disease virus was passaged 20 times by alternately adsorbing the virus with homogenized calf kidney and propagating the adsorption-resistant fraction of virus in cell cultures. Passage of the virus was accompanied by a marked decrease in pathogenicity for 35-day-old mice and a reduction in its ability to adsorb to homogenized kidney from such mice. Adsorption by homogenized calf kidney and infant mouse kidney also decreased with passage, but pathogenicity for cattle and infant mice remained high. The passaged virus did not seem to change antigenically. When the 20th-passage virus was serially passaged 10 times in cell cultures, no reversion in pathogenicity for 35-day-old mice was observed.
Inhibition of
3
H-Uridine Uptake in Mouse Macrophages Infected with
Mycobacterium lepraemurium
Authors: Ozato Keiko , Oiwa Koji
Published: January 2020

In mouse macrophages cultivated in vitro and infected with
Mycobacterium lepraemurium
,
3
H-uridine uptake was comparable to that in uninfected controls when cells were labeled immediately after infection but was markedly inhibited 2 to 3 hr later.
Inactivation of the Hemagglutinins of Type A Influenza Viruses by Physical and Chemical Means: an Aid to Classification
Authors: Cleeland Roy , Grunberg E. , Prince Herbert N.
Published: January 2020

The effects of temperature and treatment with sodium dodecyl sulfate, Tween 20, dithiothreitol, trypsin, or guanidine on the hemagglutinating capacity of six strains of type A influenza virus (A
0
/PR8/34, A
1
/CAME/46, A
2
/J305/57, A
2
/Bethesda/63, A
2
/HK/Aichi/68, and A
2
/HK/80/68), one strain of swine virus (A/Swine/76/?), and one equine strain (A/Equi-2/63) were determined. The two Hong Kong strains could be readily distinguished from the earlier A
2
strains by the resistance of their hemagglutinins to trypsin treatment and their inability to recover hemagglutinating capacity after removal of dithiothreitol from treated virus preparations. In these respects, the equine strain most closely resembled the Hong Kong variants. The pattern of hemagglutination inactivation also set the swine, PR8, and CAME strains apart from each other as well as from the other five strains. The results suggest that separation of type A viruses into groups by the pattern of inactivation of their hemagglutinins may be a valuable adjunct to standard serology for a more definite classification of these viruses.
Efficiency of Plating on Chick Embryo Cells and Kinetic Neutralization of
Herpesvirus hominis
Strains
Authors: Wentworth Berttina B. , Zablotney Sharon L.
Published: January 2020

Since it appeared that plaque formation in monolayers of primary chick embryo cells might provide a simple technique for the typing of
Herpesvirus hominis
strains, 100 isolates were tested for their efficiency of plating (EOP) on chick embryo cells versus plating on human embryonic fibroblasts. EOP values varied from 10
0
to 10
−6
: 88% of the strains of genital origin had an EOP equal to or greater than 10
−2
, and 82% of the oral isolates had an EOP equal to or less than 10
−3
. Kinetic neutralizations were done with 53 strains, including those 12 with an EOP of 10
−2
or 10
−3
. An estimate of antigenic relatedness (R
a
) between strains was calculated from the neutralization results. Although the site of recovery, EOP, and R
a
generally correlated, the EOP of some oral strains did not agree with the neutralization results, and some genital strains showed type 1 EOP and R
a
values. Selection of variants with increased EOP values did not result in accompanying changes in R
a
. Thus, the two markers appeared to vary independently. These data support other findings which suggest that there may be no absolute correlation between biological and antigenic markers in herpesviruses and that a larger number with more diversity of strains should be examined for more markers before a typing system is established.
Alkaline and Acid Phosphatase in Murine Leukemia
Authors: Wilson Donald A. , Sharma Narsingh N. , Brown Eric R.
Published: January 2020

Alterations for acid and alkaline phosphatase levels and their pattern of splenic and lymph node activity in normal and virus-induced lymphoblastic leukemia were studied. Enzyme levels were examined by using both cytochemical and biochemical procedures. The GC leukemia virus, a ribonucleic acid murine virus antigenically related to the Rauscher-Moloney viruses, was used to stimulate acid and alkaline phosphatase by producing lymphomaceous disease in Ha/ICR mice. With the Burstone and Gomori cytochemical procedures, both enzymes were found in higher than normal levels in lymphomaceous spleen and lymph nodes. Confirmation of the cytochemical studies was obtained by enzyme assay of cell-free homogenates in each case with the exception of spleen acid phosphatase. The discrepancy between the cytochemical tests which showed significant elevation of spleen acid phosphatase and the enzyme assays which failed to reveal such elevation could be due to a labile acid phosphatase isozyme which is lost on cellular disruption during homogenate preparation. A significant spleen alkaline phosphatase specific activity elevation above normal was found with a 50% incidence only when leukemic spleen wet weight increased nearly threefold its normal value. This result suggests that alkaline phosphatase elevation is a secondary event occuring after the onset of disease and is not a fundamental metabolic alteration concerned with the onset of murine lymphoblastic leukemia.
Agglutination of
Staphylococcus aureus
by Rabbit Sera
Authors: Forsgren Arne , Forsum Urban
Published: January 2020

Of 137
Staphylococcus aureus
strains, 87 agglutinated in normal rabbit serum. The agglutination was shown to be caused by the Fc-part of immunoglobulin G (IgG). F(ab
1
)
2
-fragments of IgG and immunoglobulin M (IgM) in corresponding concentrations were unreactive. The agglutinating strains had a high or moderate content of protein A. Strains with a low content of protein A and protein A-negative mutants did not agglutinate. The importance of the reaction between the Fc part of IgG and protein A for serotyping of
S. aureus
is demonstrated. Two alternative methods for serotyping
S. aureus
are suggested, using either F(ab
1
)
2
fragments of IgG or intact IgM.
Serum and Nasal Secretion Immune Response in Meningococcal Disease
Authors: Wenzel Richard P. , Mitzel John R. , Davies John A. , Beam Walter E.
Published: January 2020

Nasal antibodies to meningococcal organisms were demonstrable by the indirect fluorescent-antibody test in three patients. Serogroup cross-reactions were usual.
Antigenic Cross-Reactivity of Staphylococcal Enterotoxins
Authors: Johnson Howard M. , Bukovic Joann A. , Kauffman Peter E.
Published: January 2020

The antigenic cross-reactivity of staphylococcal enterotoxins types A, B, and C was assessed using anti-A and anti-B antitoxins in the solid-phase radioimmunoassay test. Heterologous reactions were observed. At the 33% inhibition level, B was 18,000 and 5,400 times more effective as an inhibitor in its homologous system than were the heterologous enterotoxins A and C, respectively. Similarly, in the A system, A enterotoxin was 55,000 and 25,000 times more effective than were B and C toxins, respectively, in inhibiting A-anti-A reactions.
Experimental Studies on Cholera Immunization II. Evidence for Protective Antitoxic Immunity Mediated by Serum Antibodies as Well as Local Antibodies
Authors: Holmgren J. , Andersson Å. , Wallerstrom G. , Ouchterlony Ö.
Published: January 2020

By use of the ileal loop technique, the resistance to challenge with cholera enterotoxin was compared between unimmunized rabbits and rabbits immunized with toxin or toxoids. It was shown that subcutaneous as well as intraintestinal immunization induced protective immunity, the toxin being a better immunogen than Formalin-induced toxoid and much better than heat-induced toxoid. The relation between protection and serum antitoxin titer was poor, e.g., protection was seen in the absence of demonstrable serum antibodies. However, intravenous administration of antitoxic antiserum conferred some protection, suggesting that local as well as serum-mediated antitoxic immunity is operating in the host defence against cholera.
Correlation of M Protein Production with Those Factors Found to Influence Growth and Substrate Utilization of
Streptococcus pyogenes
Authors: Pine Leo , Reeves Michael W.
Published: January 2020

In the absence of proteinase formation, factors reported to influence the growth or fermentation by streptococci have been evaluated to determine their quantitative effect upon the production of M protein during the growth of
Streptococcus pyogenes.
Buffers, amino acids, peptides, gross organic additions, and carbohydrate substrates were tested under a variety of cultural conditions. The M protein content was remarkably constant throughout the late logarithmic period of growth, i.e., when the cell population doubled, the M protein doubled. However, several factors affected the M protein content per milligram of cells (dry weight). When types 1, 12, and 22 were grown aerobically in a semidefined medium, the M protein content of the cell population essentially doubled; in Todd-Hewitt broth, this aerobic effect on M protein synthesis was not observed. When cells grown on Todd-Hewitt broth were transferred to medium containing 0.1% starch and no added glucose, the M protein content per milligram of cells (dry weight) increased as much as fourfold. When growth was initiated in glucose, the rate of M protein formation was at a maximum in the early logarithmic phase of growth and was comparatively greater than the rate of cellular multiplication. When the amount of substrate fermented was greater than 0.2%, increased M protein was not observed. An evaluation of the effects of medium or conditions of growth showed the units of M per milligram of cells (dry weight) were not influenced by a shift in the stoichiometry of either the anaerobic or aerobic fermentation, substrate used, or adenosine triphosphate utilized for growth. These results show that M protein synthesis is subject to limited glucose repression or substrate catabolite repression.
Staphylococcidal Capability of Rabbit Peritoneal Macrophages in Relation to Infection and Elicitation: Delayed-Type Hypersensitivity Without Increased Resistance
Authors: Lenhart Nancy , Mudd Stuart
Published: January 2020

The staphylococcidal capability of populations of peritoneal macrophages in rabbits has been measured before and after repeated infections with
Staphylococcus aureus.
Such rabbits after infection showed delayed-type hypersensitivity to
S. aureus
antigens, but the staphylococcidal capability of the peritoneal macrophages was not increased. This result at the cellular level is in agreement with previous assessment in vivo of the consequences of staphylococcal infection. Pathways to cell-mediated resistance, with and without delayed-type hypersensitivity, are presented.
Mutation in
Shigella flexneri
2a Resulting in Increased Electrophoretic Mobility
Authors: Corwin Laurence M. , Talevi Leonard A.
Published: January 2020

An avirulent mutant of
Shigella flexneri
2a, when grown in broth containing calcium ions, was found to have a three- to fourfold increase in electrophoretic mobility toward the anode when compared to its virulent parent. Under these same growth conditions, it was found that the avirulent strain was made more electronegative in the presence of ethylenediaminetetraacetic acid (EDTA). EDTA had no effect on the electrophoretic mobility of the virulent strain. In the absence of added calcium in the growth medium, no differences between the strains were observed. The alteration which has resulted in an increased negative charge has also stabilized the avirulent strain against loss of lipopolysaccharide from EDTA treatment.
Mechanisms of Pathogenesis in
Listeria monocytogenes
Infection V. Early Imbalance in Host Energy Metabolism During Experimental Listeriosis
Authors: McCallum R. E. , Sword C. P.
Published: January 2020

Early changes in hepatic carbohydrate metabolism without apparent hepatocyte dysfunction were reported previously in mice infected with
Listeria monocytogenes.
This study was undertaken to examine possible imbalance in host regulatory mechanisms which might be responsible for these changes. Female CD-1 mice fasted 12 hr prior to the experiments were injected intraperitoneally with 10
5
, 10
6
, or 10
7
Listeria.
Control mice received either 10
9
heat-killed
Listeria
or 150 μg of
Salmonella typhimurium
lipopolysaccharide. Hepatic glycogen, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and nicotinamide adenine dinucleotide (NAD) (NAD
+
, NADH, NADP
+
, and NADPH) levels were assayed periodically. Activities of ATP hydrolyzing enzyme and NAD glycohydrolase were measured at various intervals after infection. Decreases in glycogen occurred as early as 10 hr after infection. Responses in the controls differed from those in infected mice. Hepatic ATP levels decreased as early as 10 hr after infection, with concomitant increases noted in ADP. Hepatic ATP hydrolyzing enzyme activity increased as the infection progressed. Decreases were noted in hepatic NAD levels, with the greatest reduction in the reduced form of NAD. Slight changes were observed after 10 hr, and greater differences were noted 20 hr after infection. The magnitude of these biochemical changes appeared to be dose-dependent. Significant increases in hepatic NAD glycohydrolase activity were noted as the infection progressed. Small but significant increases in serum inorganic phosphate were noted 10 and 20 hr after infection, with a larger increase observed 30 hr after infection. The results indicate impairment of host energy metabolism early in the course of experimental listeriosis.
Immunological Unresponsiveness Induced by Cryptococcal Capsular Polysaccharide Assayed by the Hemolytic Plaque Technique
Authors: Murphy Juneann W. , Cozad George C.
Published: January 2020

Numerous studies have suggested that cryptococcal capsular polysaccharide could induce an immunological paralysis. To investigate this possibility, mice were given various concentrations of purified cryptococcal polysaccharide and then 14 days later were challenge-immunized with the same material in Freund's incomplete adjuvant. Anticryptococcal agglutinin titers were determined at various periods after polysaccharide treatment and after challenge immunization. At the same periods the hemolytic plaque technique was used to determine the number of spleen cells capable of producing antibody against cryptococcal polysaccharide. The data indicated that there was a transitory immune response which preceded tolerance induction. In animals given the largest doses of polysaccharide, “in vivo” neutralization was responsible for low serum agglutinin titers during the transitory response. The capsular polysaccharide was considered to have induced immunological unresponsiveness at the highest concentration, because challenge immunization did not stimulate an increase in the number of plaque-forming cells (PFC). A sixfold increase in numbers of PFC was found in animals injected initially with the lowest concentration of polysaccharide. These results support the idea that tolerance was due to terminal differentiation without proliferation of the immunocompetent cells. The central failure of the immune mechanism which was apparent in the paralyzed mice was temporary under the conditions of this experiment.
Experimental Infection of the Skin in the Hamster Simulating Human Impetigo IV. Cellular Responses after Streptococcal and Staphylococcal Infections
Authors: Dajani Adnan S. , Wannamaker Lewis W.
Published: January 2020

Various cellular responses to skin infections in an experimental animal model were explored. Total leukocyte counts varied after group A streptococcal infections, but a depression was commonly seen after M type 12 impetigo.
Staphylococcus aureus
infections resulted in moderate leukocytosis. A marked neutrophilia was universal with streptococcal or staphylococcal disease. A positive nitroblue tetrazolium (NBT) response appeared 24 hr after infection, reached a peak in 48 hr, and then declined. This occurred in the absence of extensive cellulitis or bacteremia. An increase in the percentage and absolute number of NBT-positive neutrophils occurred. M type 57 streptococcus produced a more strongly positive NBT test than did M type 12. Cell-free filtrates of a broth culture of M type 57 streptococcus produced NBT responses in hamsters comparable to the responses seen after injection of live organisms. These studies indicate the usefulness of this animal model to study various parameters of the NBT test.
Isolation of the Outer Sheath of
Leptospira
and Its Immunogenic Properties in Hamsters
Authors: Auran N. E. , Johnson R. C. , Ritzi D. M.
Published: January 2020

Spherical forms of
Leptospira interrogans
serotype
canicola
Hond Utrecht IV were induced with 1
m
NaCl. Electron micrographs of these salt-altered cells (SAC) revealed that the outer envelope or sheath had pulled away from the protoplasmic cylinder. Treatment of SAC with 0.02% sodium lauryl sulfate solubilized the sheath and released the protoplasmic cylinder. Further processing of the solubilized sheath yielded a pellet which displayed a membrane structure in electron micrographs. The released protoplasmic cylinder showed loss of intracellular organization and the outer envelope present in normal cells. Immunization of hamsters with whole formalized cells, SAC, or sheath in doses as low as 10 μg/animal protected them from death upon challenge with virulent
canicola
27.
Antigenic heterogeneity of outer membrane proteins of nontypable Haemophilus influenzae is a basis for a serotyping system
Authors: Murphy T F , Apicella M A
Published: January 2020

A serotyping system for nontypable Haemophilus influenzae (NTHI) was developed by using isolated outer membrane protein (OMP) preparations and rabbit antisera. OMPs of 23 strains were isolated by molecular sieve chromatography of outer membranes in 1.5% sodium deoxycholate buffer. These OMP preparations were relatively free of lipopolysaccharide as determined by silver staining of sodium dodecyl sulfate gels and by dot assay with a monoclonal antibody which is specific for the lipid A of H. influenzae. Three antisera raised to whole organisms were used to serotype 21 of 23 strains with a kinetic enzyme-linked immunosorbent assay. Digestion of OMP preparations with proteinase K removed greater than 90% of the antigenic reactivity, indicating that the system is based on OMP antigens. Marked antigenic heterogeneity of OMPs exists among strains of NTHI. By determining the pattern of serological reactivity of OMPs with the three antisera, isolates were divided into groups based on antigenic differences. Six serotypes were identified. This OMP serotyping system is based on multiple antigenic determinants. Future studies will focus on identifying serotype-specific epitopes to further refine this serological classification scheme for NTHI.
Role of C5 and recruited neutrophils in early clearance of nontypable Haemophilus influenzae from murine lungs
Authors: Toews G B , Vial W C , Hansen E J
Published: January 2020

We used a mouse model system to investigate the pulmonary defense mechanisms involved in clearance of nontypable Haemophilus influenzae from the lower respiratory tract. The importance of the C5 complement protein molecule in polymorphonuclear leukocyte (PMN) recruitment was studied by using congenic C5-sufficient B10.D2/nSn (C5+) and C5-deficient B10.D2/oSn (C5-) mice. The C5+ and C5- mice were inoculated with saline or nontypable H. influenzae via an endobronchial catheter. Clearance of bacteria was studied by using quantitative lung cultures. Bronchoalveolar lavage was performed at several time intervals. The number of cells in the lavage fluid were counted, and chemotactic activity was assayed in lavage fluid by the leading front technique, using human PMN in modified Boyden chambers. Pulmonary clearance of bacteria was significantly impaired in the absence of C5 (P less than 0.05). The C5+ mice recruited significantly more PMN after challenge with nontypable H. influenzae than C5- mice did (P less than 0.05), but significant PMN recruitment occurred in C5- mice. Similarly, although chemotactic activity was present in both C5+ and C5- mice, significantly more intraalveolar chemotactic activity was noted in C5+ mice than in C5- mice (P less than 0.05). The C5 molecule yields important chemotaxins during this early time period, but other chemotaxins are also present within the alveoli, demonstrating the redundancy of the inflammatory response after pulmonary challenge with nontypable H. influenzae. Nitrogen mustard-induced neutropenic animals were studied to evaluate the functional importance of PMN in pulmonary clearance of nontypable H. influenzae. Pulmonary clearance of nontypable H. influenzae was significantly impaired in neutropenic animals (P less than 0.05). Our results indicate that the prompt appearance of PMN in lungs is crucial for early clearance of nontypable H. influenzae.
Cloning and surface expression in Escherichia coli of a structural gene encoding a surface protein of Haemophilus influenzae type b
Authors: Holmans P L , Loftus T A , Hansen E J
Published: January 2020

Recombinant DNA technology was used to clone a gene coding for a surface protein of Haemophilus influenzae type b (Hib) into Escherichia coli. Chromosomal DNA from a clinical isolate of Hib was cleaved with EcoRI and ligated into plasmid vectors containing three different translational reading frames. E. coli carrying recombinant plasmids were screened in a colony blot-radioimmunoassay system by using murine monoclonal antibodies (mabs) directed against cell surface-exposed proteins of Hib. mab 7B2, which is specific for a Hib surface protein with an apparent molecular weight of 27,000 (27K), reacted with several recombinant strains of E. coli. Restriction analysis revealed the presence of a 9.1-kilobase DNA insert in each of these recombinant plasmids and also determined that both transcription and translation of the Hib gene(s) coding for the 7B2-reactive antigen were not dependent on the lac operator and promoter of the vectors. Radioimmunoprecipitation and Western blot analyses showed that the antigenic determinant recognized by mab 7B2 in these recombinant E. coli was present in a 27K protein. In addition, this 27K protein was shown to be both localized on the surface of these E. coli cells and accessible to antibody.
Phagocytosis of staphylococci by human polymorphonuclear leukocytes is enhanced in the presence of endothelial cells
Authors: Vandenbroucke-Grauls C M , Thijssen H M , Verhoef J
Published: January 2020

The role of various surfaces in the phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) was studied. Uptake of both opsonized and unopsonized staphylococci on the surface of a monolayer of human venous endothelial cells was compared with uptake on an inert plastic surface, with an assay that uses radiolabeled bacteria. Uptake of unopsonized S. aureus was threefold higher on the endothelial cell surface than on the plastic surface and was followed by efficient killing of the phagocytosed staphylococci. Uptake of unopsonized S. aureus on endothelial cells was not inhibited by treatment of the PMN with pronase or 2-deoxy-D-glucose and was only partially inhibited by cytochalasin B treatment of the PMN. The supporting effect of endothelial cells on the phagocytosis of unopsonized S. aureus was not due to opsonization of the bacteria by immunoglobulin or complement from the endothelial cell surface, nor to coating with fibronectin.
Experimental enterotoxin-induced Escherichia coli diarrhea and protection induced by previous infection with bacteria of the same adhesin or enterotoxin type
Authors: Ahrén C M , Svennerholm A M
Published: January 2020

The diarrheal response to an initial and a second infection with Escherichia coli expressing various enterotoxins (the heat-stable toxin [ST] alone or in combination with the heat-labile toxin [LT]) and colonization factor antigens (CFA/I, CFA/II, or E8775-type) was studied in the reversible tie adult rabbit diarrhea model. An initial infection with high doses (1 X 10(10) to 5 X 10(11) bacteria) of the various strains regularly induced diarrhea which was usually self-limiting (only 7 of 85 animals died). The diarrheal response to equally effective doses of different strains producing both ST and LT (ST/LT) did not differ significantly with serotype or colonization factor antigen. ST/LT-producing strains appeared to induce severe disease more regularly than ST-producing strains carrying the same adhesin. Previous infection with CFA/I-carrying, ST/LT-producing E. coli protected all animals reinfected with an otherwise highly diarrheogenic dose of the same strain as well as against challenge with a CFA/I-carrying, ST/LT-producing strain with different O-, K-, and H-antigens. Fecal excretion of bacteria was also significantly reduced in the protected animals, although not completely eliminated. When only one of the two antigens, CFA/I and LT, was shared by the immunizing and rechallenge strains, partial protection was evident consistent with independent antibacterial (anti-CFA) and antitoxic (anti-LT) immune mechanisms. Oral immunization with purified CFA/I significantly reduced fluid secretion in intestinal loops infected with CFA/I-carrying enterotoxigenic bacteria.
Functional role of interleukin 1 in periodontal disease: induction of interleukin 1 production by Bacteroides gingivalis lipopolysaccharide in peritoneal macrophages from C3H/HeN and C3H/HeJ mice
Authors: Hanazawa S , Nakada K , Ohmori Y , Miyoshi T , Amano S , Kitano S
Published: January 2020

Hot phenol-water-extracted lipopolysaccharide (LPS) from Bacteroides gingivalis 381 was purified by Sephadex G-100 chromatography with Tris buffer supplemented with sodium deoxycholate and EDTA (B-LPS). In the present study, B-LPS was examined for its ability to induce interleukin 1 (IL-1) production, a mitogenic response, and macrophage activation in LPS high-responder C3H/HeN and low-responder C3H/HeJ mice. A significant increase in IL-1 production was observed in C3H/HeN and C3H/HeJ peritoneal macrophages treated with various doses (1.0 to 50 micrograms/ml) of B-LPS. IL-1 production by C3H/HeN macrophages treated with B-LPS (10 micrograms/ml) was about seven times greater than that by C3H/HeJ macrophages. However, the IL-1 production induced by B-LPS (10 micrograms/ml) in C3H/HeN macrophages was four times lower compared with that induced by Escherichia coli O111 B4 LPS. Also, a significant increase in IL-1 production was found in human monocytes stimulated with B-LPS. That B-LPS-induced IL-1 exhibits some molecular weight heterogeneity was indicated from Sephadex G-75 gel filtration profiles. A significant, high mitogenic response by whole spleen cells with 1 X 10(5) to 5 X 10(4) cells of either mouse strain per well treated with B-LPS (10 to 50 micrograms/ml) was observed. However, the response of C3H/HeJ mice was less than that of the C3H/HeN strain. Also, glucose consumption assays indicated that enhanced macrophage activation occurred in C3H/HeN but not in C3H/HeJ mice treated with B-LPS. In light of recent studies showing that IL-1 stimulates bone resorption in a mouse calvaria system and collagenase production in fibroblasts, we suggest that B-LPS-induced IL-1 may play a significant role in the pathogenesis of adult periodontal disease.
Basis for serological heterogeneity of thermostable antigens of Campylobacter jejuni
Authors: Mills S D , Bradbury W C , Penner J L
Published: January 2020

Lipopolysaccharides (LPS) were extracted from eight strains of Campylobacter jejuni and purified by enzyme treatment to remove traces of RNA, DNA, and protein. This material was used to sensitize sheep erythrocytes for the passive hemagglutination assay that is presently used to serotype C. jejuni. The results confirmed that the thermostable antigen typing scheme is based on LPS (O) antigens. The LPS after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining was found to consist of a series of slow migrating bands which could not be eliminated by treatment with NaOH, urea, or EDTA. However, the use of LPS double labeled with 14C and 32P yielded evidence that the bands of high molecular weight were indeed aggregations of low-molecular-weight LPS molecules.
Actinlike material in Pseudomonas aeruginosa
Authors: Berk R S , Moon M , Higbee R , Cramer C , Roher A , Weis M , Hazlett L
Published: January 2020

Actinlike material was obtained from disrupted Pseudomonas aeruginosa cells by a modification of the method of Hancock and Nikaido (J. Bacteriol. 136:381-390) for isolating outer membrane vesicles. Pelleted membranes were dissolved in Laemmli sample buffer and electrophoresed in parallel lanes with purified rabbit skeletal muscle actin. The bacterial preparation migrated similarly to rabbit skeletal muscle actin. A doublet band was detectable by affinity-purified antiactin antibody in a passive transfer immunoblot. Molecular weight of the actinlike protein doublet was 60,000 to 63,000 as determined by linear regression analysis of Bio-Rad low-molecular-weight standards run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transmission electron microscopy of the actinlike material in high salt concentrations revealed 10- to 14-nm filaments of various lengths. Despite its ability to form filaments and to react with a polyclonal rabbit antiactin antibody, the bacterial filaments did not bind the S-1 fragment of heavy meromyosin.
Differences in outer membrane proteins of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis
Authors: Batteiger B E , Newhall W J , Jones R B
Published: January 2020

The lymphogranuloma venereum (LGV) and trachoma biovars of Chlamydia trachomatis exhibit differences in biological properties both in vivo and in vitro. To identify analogous biochemical differences, we studied the molecular charges of chlamydial outer membrane proteins (OMPs) by means of isoelectric focusing and nonequilibrium pH gradient electrophoresis. Analysis of proteins of whole elementary bodies biosynthetically labeled with L-[35S]cysteine revealed that most chlamydial proteins were neutral or acidic. The major OMPs (MOMPs) of all strains tested were acidic and had apparent isoelectric points (pIs) that varied within narrow limits (approximately 5.3 to 5.5) despite differences in molecular mass of up to 3,000 daltons (Da). However, a low-molecular-mass cysteine-rich OMP analogous to that previously described for Chlamydia psittaci varied consistently in molecular mass (12,500 versus 12,000 Da) and pI (5.4 versus 6.9) between LGV strains and trachoma strains, respectively. OMPs with a molecular mass of 60,000 Da in the trachoma biovar strains had pIs in the 7.3 to 7.7 range. However, analogous OMPs in the LGV strains existed as a doublet with a molecular mass of about 60,000 Da. Both members of the doublet were basic (pIs greater than 8.5). Both proteins of this basic doublet in LGV strains and the neutral analog in trachoma strains bound a species-specific monoclonal antibody in an immunoblot assay. These data indicate substantial differences in biochemical characteristics of analogous OMPs in the LGV and trachoma biovars. Such differences are the first structural differences described between LGV and trachoma strains which support their distinction into separate biovars and may be related to some of their biological differences.
Plasmid-cured Salmonella enteritidis AL1192 as a candidate for a live vaccine
Authors: Nakamura M , Sato S , Ohya T , Suzuki S , Ikeda S , Koeda T
Published: January 2020

We report the immunizing capacity of Salmonella enteritidis AL1192, a strain that has been cured of a 36-megadalton plasmid, to protect ddY mice against subsequent challenge with virulent salmonellas. This strain, which was given subcutaneously at a dose of 10(6) organisms, provided significant protection against oral, subcutaneous, or intraperitoneal challenge by virulent wild-type strains of not only S. enteritidis, but also S. dublin, S. naestved, and S. typhimurium.
Characterization of the insertion of Pseudomonas exotoxin A into membranes
Authors: Zalman L S , Wisnieski B J
Published: January 2020

Pseudomonas aeruginosa exotoxin A (PTx) is an extremely potent inhibitor of protein synthesis, similar to diphtheria toxin in its mode of action. It is synthesized in precursor form and secreted as an Mr 66,583 protein lacking a 25-amino acid leader sequence. While the primary sequence and the nature of the enzyme activity that leads to inactivation of elongation factor 2 are known, the mechanism of PTx internalization remains obscure. To elucidate the entry pathway, we examined PTx-membrane interactions using vesicle targets of defined lipid composition. Insertion was monitored with an intramembranous photoreactive probe; pore formation was determined from liposomal swelling rates. Our results show that the efficiency of PTx binding to vesicles increases dramatically with decreasing pH. In general, the insertion efficiency correlated with the binding efficiency. At pH 4, we noted a slight decrease in binding below the melting point (23 degrees C) of the target vesicles. Not only was PTx able to insert into frozen bilayers, but the efficiency of penetration at 0 degrees C was actually somewhat higher than expected based on binding efficiency. Liposome swelling assays analyzed by the Renkin equations indicated that PTx-liposomes made at pH 4 were permeable to solutes up to 2.8 nm in diameter. Pores of a similar size were found when the liposomes were made at pH 7, but the efficiency of pore formation at this pH was very low. Chymotrypsin fragmentation profiles of PTx depended on incubation conditions, e.g., pH, presence of NAD, reducing agents, and membranes. Liposomes containing PTx cleaved at pH 4 displayed up to 40-fold more pore activity than liposomes containing uncleaved PTx or PTx cleaved at pH 7. Pore activity at pH 7 was negligible. Addition of reducing agents caused a 50 to 60% increase in pore activity. Cleaved toxin was active in target membrane insertion even at 0 degrees C, and all of the major fragments were photolabeled.