Human monoclonal antibodies to glycolipid A that exhibit complement species-specific effector functions.

Two human IgM monoclonal anti-glycolipid A antibodies (MAbs) were evaluated for their abilities to bind to various endotoxins and pathogenic gram-negative bacteria and to activate complement pathways, thereby accomplishing bactericidal and opsonic effector functions. Both MAbs cross-reacted with glycolipid A mutant lipopolysaccharides from rough colony-forming gram-negative bacteria and with selected endotoxins from smooth colony-forming bacteria. However, MAb 10235 bound to all clinical isolates of Escherichia coli and Klebsiella pneumoniae tested but only very weakly to Pseudomonas aeruginosa, whereas MAb 10058 bound to all three genera. Several strains of serum-insensitive organisms were selected for evaluation of antigen-specific, complement-mediated effector functions for the two MAbs. Assessment of bactericidal and opsonic activities showed that neither MAb was able to activate complement from nonprimate species (mouse, rat, rabbit, guinea pig, or sheep). However, both MAbs were highly effective in using primate sources of serum complement to mediate these effector functions.

Human monoclonal antibodies specific for capsular polysaccharides of Klebsiella recognize clusters of multiple serotypes.

We report the generation and the characterization of a set of human monoclonal antibodies (HmAb) specific for Gram-negative bacteria of Klebsiella pneumoniae. The eight human hybridomas secrete either IgM kappa, IgA1 kappa, or IgA2 kappa antibodies. One HmAb binds bacteria of only one serotype. Five HmAb recognize non-overlapping clusters of 2, 3, or 10 different serotypes. The remaining two HmAb both recognize three serotypes. Two serotypes are recognized by both HmAb, and in addition both HmAb bind one more nonidentical serotype. These results suggest that in man, epitopes are immunodominant, different from serotype-specific determinants detected by conventional rabbit antisera. Screening of clinical isolates revealed that the HmAb recognize not only representative typing strains but also most isolates of the corresponding serotype. In addition, most of the isolates that were non-typable by polyclonal antisera were recognized by one of the HmAb. Fine specificity analyses revealed that all HmAb are highly specific for the isolated capsular polysaccharides (CPS) of bacteria within the corresponding cluster of serotypes. However, the avidity of a HmAb for the different CPS can differ significantly. Taken together, our results suggest that the unequivocal interactions between HmAb and CPS offer the basis for an alternative, better defined classification system, and that passive immunization with a limited number of HmAb may provide a feasible strategy for the protection against the majority of fatal, nosocomial infections with multidrug-resistant strains of K. pneumoniae.

Systematic generation of antigen specific human monoclonal antibodies with therapeutical activities using active immunization.

Presenting a panel of human hybridomas secreting serospecific antibodies which confer a high degree of protection against fatal infection with Pseudomonas aeruginosa, we report an efficient approach for a systematic generation of antigen specific human monoclonal antibodies with biological activity. This approach is based on active immunization and antigen specific panning. Individuals were immunized with polysaccharides isolated from LPS of Pseudomonas aeruginosa conjugated to toxin A. Specific B cells were isolated and enriched by panning of blood samples taken at the time point with the highest frequency of fuseable cells. These cells were then transformed with Epstein-Barr virus. Arising lymphoblastoid cell lines were screened for the secretion of anti-LPS antibodies by enzyme-linked immunosorbent assay and fused to a murine-human heteromyeloma cell line. Hybridomas were selected for high levels of antibody secretion and binding to intact bacteria as determined by an immunofluorescence microscopy assay. The observation that protective capacity of an antibody was associated with its ability to bind to LPS determinants accessible on the bacterial cell surface allowed for an effective screening for therapeutically interesting human monoclonal antibodies. Out of four immunized individuals, 15 lymphoblastoid cell lines with anti-LPS activity could be isolated, and 8 hybridomas, which cover the majority of the common Pseudomonas aeruginosa serotypes, were characterized further. The generation of monoclonal anti-Pseudomonas aeruginosa toxin A, anti-Klebsiella capsular polysaccharides, and anti-Escherichia coli LPS antibodies suggests that the success of this approach is not limited to the generation of human monoclonal antibodies of a particular specificity or to the use of antigens of a particular character.

Influence of bovine mastitis on lipolysis ond proteolysis in milk.

Lipolysis and proteolysis in milk were determined before, during, and after experimentally induced mastitis. Streptococcus agalactiae was infused into one quarter of five cows to elicit an infection. Milk protease activity was higher during infection, but milk lipase activity was unchanged. Lipolytic damage to milk fat and proteolytic damage to milk casein occurred in the udder prior to milking during an infection. Lipolysis increased due to increased susceptibility of the milk fat to lipase action during infection. The mechanism of the increased susceptibility of the fat to lipolysis was not determined. After infections were eliminated, SCC, initial and stored FFA concentrations, and initial tryosine values returned to preinfection levels. However, after infections were eliminated, milk protease activity as determined by an increase in tryosine values remained elevated as milk SCC returned to preinfection levels. Protease activity returned to preinfection levels within 10 d after SCC returned to preinfection levels.

Selection of tests for monitoring the bacteriological quality of refrigerated raw milk supplies.

Raw milk samples were collected from 10 producer bulk tanks. Samples were then subdivided so that milks were subsequently stored at 1.7, 4.4, 7.2, and 10.0 degrees C for 24 and 48 h. After storage, samples were analyzed by seven plating methods: standard plate count, psychrotrophic bacterial count, rapid psychrotrophic count, preliminary incubation count, mesophilic plate count, laboratory pasteurized count, and coliform count by violet red bile agar technique. Impedance protocols on a Bactometer Model 123 for total count, psychrotrophic count, mesophilic count, and coliform count were also used to evaluate the bacteriological quality of the milks. Bacterial counts and impedance detection times were analyzed using nonparametric statistics. Impedance protocols for total count and psychrotrophic count were the best indicators of bacteriological quality. Preliminary incubation count was the best of the plating methods. The laboratory pasteurized count performed poorly. Impedance measurements provided information in the shortest time.

Comparison of dry culture medium and conventional plating techniques for enumeration of bacteria in pasteurized fluid milk.

Standard plate counts, psychrotrophic bacterial counts, and coliforms were determined by conventional plating techniques and by Petrifilm TM plates, a dry culture medium, for 48 commercially processed milk samples (24 whole milk and 24 skim milk). The Petrifilm SM plate counts were compared with counts on standard methods agar for the standard plate count, psychrotrophic bacterial count, and rapid psychrotrophic bacterial count. The Petrifilm violet red bile plate counts were compared with counts on violet red bile agar for coliform test with a solid medium and the preliminary incubation method for detection of coliforms. Standard plate counts were determined within 24 h of packaging and after 7, 10, and 14 d of storage at 6.1 degrees C. Psychrotrophic bacterial counts and coliform counts were determined with 24 h of packaging and after 7 d storage. There was a strong linear relationship between Petrifilm SM and standard methods agar plates (excluding counts on samples plated within 24 h of packaging) and for the psychrotrophic bacterial count method. Petrifilm SM had a weak linear relationship with Standard Methods Agar plates for the rapid psychrotrophic bacterial count. Coliform counts determined on Petrifilm violet red bile plates were generally within the same range as counts on violet red bile agar plates. The positive predictive values for the Petrifilm violet red bile plates and violet red bile agar plates were essentially the same for samples plated within 24 h of packaging.

Recombinant tumor necrosis factor causes activation of human granulocytes.

We have tested the hypothesis that tumor necrosis factor (TNF), by binding to and activating granulocytes, may contribute to the pathogenesis of gram-negative sepsis and the adult respiratory distress syndrome (ARDS). Buffy coat granulocytes incubated with as little as 0.5 ng/mL of recombinant TNF (rTNF) showed a dose-related increase in nitroblue tetrazolium dye reduction, in granulocyte polarization, in superoxide anion release, and in visually apparent aggregation. Purified lipopolysaccharide (1 microgram/mL) caused polymorphonuclear (PMN) aggregation and activation that was neutralized by polymyxin B. The release of superoxide was augmented by preincubation of the PMNs with gamma-interferon. The effect of TNF was neutralized by TNF-specific murine monoclonal antibodies but not by polymyxin B. Scatchard analysis of 125I-rTNF binding to granulocytes revealed about 1,200 receptors per cell with a Kd of 4.9 X 10(-10) mol/L. These results suggest that the release of TNF by mononuclear phagocytes contributes to granulocyte activation and aggregation during inflammation.

Variability in true protein, casein, nonprotein nitrogen, and proteolysis in high and low somatic cell milks.

Monthly variation in milk protein (total nitrogen X 6.38) and milk fat was determined for 115 farms over 2 yr. Yearly average milk protein and fat tests in each year were 3.16 and 3.62%, respectively. The mean regression coefficient for milk protein with respect to milk fat was .47 for the entire period. Twenty-four farms were selected and grouped high or low based on their previous 2-yr somatic cell history. Monthly milk samples for each farm were tested for direct microscopic somatic cell count, total nitrogen, noncasein nitrogen, and nonprotein nitrogen. No differences in monthly nonprotein nitrogen, true protein, and casein were found between groups. Casein as a percent of total nitrogen was significantly higher for the low somatic group for seven of the 12 mo studied but was significantly higher for 9 mo when expressed as a percent of true protein. The average increase in tyrosine value for incubated preserved milk was significantly higher for the high somatic cell milk, indicating higher proteolytic activity in high somatic cell milk. Electrophoretic analysis of high and low somatic cell milk indicated that there was substantial proteolytic breakdown of alpha S-casein and beta-casein by proteases associated with elevated somatic cell counts.

Human monoclonal antibodies against group A red blood cells.

A human monoclonal antibody with anti-A specificity was generated by Epstein-Barr virus transformation of lymphocytes isolated from splenic tissue after in vitro stimulation with group A red blood cells. This antibody is of the IgM class and directly agglutinates group A red blood cells. Type A1, A2, Aint, A3, AX, Aend and A5 cells were agglutinated by the reagent indicating a single determinant is shared by these A subgroups.

Characterization of human hybridomas secreting antibody to tetanus toxoid.

We have selected a thioguanine-resistant lymphoblastoid cell line (LTR228) that forms human-human hybrids with high efficiency. Fusions with peripheral B cells consistently yield one colony per 10(5) cells plated. To produce antitetanus monoclonal antibodies, we withdrew blood from persons who had recently received booster injections of tetanus toxoid. T cells were separated from peripheral mononuclear cells by 2-aminoethylisothiouronium bromide-induced rosette formation, given 1,500 rads (1 rad = 0.01 gray), and cultured in a 1:1 ratio with nonrosetting cells. After 3 days of pokeweed mitogen stimulation, heterokaryons were produced by a plate-fusion technique and cultured in Iscove's Dulbecco's minimal essential medium for 24 hr prior to hybrid selection. Colonies appeared after 10-14 days in hypoxanthine/azaserine supplemented medium. A direct binding enzyme-linked immunosorbent assay with specific tetanus toxoid inhibition identified positive wells. The hybridomas were cloned twice in soft agarose and by limiting dilution. The subcloned hybridomas double every 26 hr (vs. every 16 hr for LTR228) and produce 1-5 micrograms of specific IgG, kappa antibody per 10(6) cells per ml per 24 hr. All subclones (almost 200) continue to secrete antibody after 11 months of continuous culture. Twelve representative subclones have near tetraploid amounts of DNA. From hybridomas grown in 5-liter spinner flasks, milligram quantities of the IgG, kappa antibody were purified by staphylococcus protein A affinity chromatography. Specific antibody from hybridoma cultures protected mice injected with 1,000 times the LD50 of tetanus toxin. Our cell line and associated techniques should permit the production of therapeutically important human monoclonal antibodies.

Immune responses to chlamydial antigens in humans.

Antibody titer, lymphocyte stimulation and leukocyte migration inhibition with chlamydial antigens were determined repeatedly over many months on human subjects. The volunteers were retrospectively placed into four groups on the basis of clinical, laboratory and epidemiologic criteria. Group A consisted of persons with proven or probable chlamydial infection, including an illness confirmed by chlamydial isolation or seroconversion, or a clinically compatible illness with positive serologic results. Group B were sexual partners or close contacts of group A individuals. Group C were laboratory workers with prolonged exposure to viable chlamydiae or their antigens. Group D included persons of comparable age as those in groups A and B, but lacking a history of symptomatic chlamydial infection or of contact with chlamydiae. Individual cases illustrated the rise of antibody and some cell mediated immunity reactions (CMI) with active chlamydial infection. By contrast, laboratory exposure resulted in elevation of CMI but not of antibody. Statistical analysis of the results in 46 volunteers tested repeatedly indicated a strong association of specific antibody with lymphocyte stimulation, but not with leukocyte migration inhibition. Regression analysis suggested that the type of exposure markedly influenced the relationship between antibody and lymphocyte stimulation. Measurement of immunotype-specific antibody titer by microimmunofluorescence (or an equally sensitive method) remains the best laboratory indicator of past chlamydial infection. Neither antibody nor CMI can, as yet, be definitely related to resistance to re-infection in humans.

Subpasteurization Heat Treatment to Inactivate Lipase and Control Bacterial Growth in Raw Milk.

Raw milk was heat-treated under subpasteurization and suprapasteurization conditions, cooled and stored for up to 72 h at 4.4 and 6.7°C. Milk lipase activity and bacteria counts were monitored in both unheated and heated milks. Inhibition of milk lipase activity ranged from 42 to 98% for treatments of 57.2°C for 10 sec to 73.9°C for 10 sec, respectively. The logs of Standard Plate Count after 72 h of storage at 6.7°C were 6.56, 4.86, 4.31, 4.00 and 2.82 for unheated and 10-sec heat treatments at 57.2, 65.6, 73.9 and 82.2°C, respectively. Psychrotrophic Bacteria Counts were also lower in the heated milks than in the unheated milk. The logs of Psychrotrophic Bacteria Counts after 72 h of storage at 6.7°C were 6.21, 2.45, 2.27, 1.33 and 1.00 for unheated and 10-sec heat treatments at 57.2, 65.6, 73.9 and 82.2°C, respectively. Heat treatment of raw milk supplies would result in limiting action of the milk lipase system and growth of bacteria.

Cell-mediated and humoral immune responses to chlamydial antigens in guinea pigs infected ocularly with the agent of guinea pig inclusion conjunctivitis.

Cell-mediated immune response and humoral response to chlamydial antigens were investigated in guinea pigs infected with the agent of guinea pig inclusion conjunctivitis (GPIC). Pronounced cell-mediated immune response to the homologous antigen, as well as to two other chlamydial antigens, 6BC (Chlamydia psittaci) and LB-1 (C. trachomatis), occurred in all infected animals. Cell-mediated immune response to GPIC, and to a lesser extent to 6BC and LB-1 as well, was enhanced with time after infection even without the re-inoculation of the infectious agent. Extensive cross-reactions among the three chlamydial antigens during the cell-mediated immune response appeared to be due to shared species-specific and group-reactive antigens. Serum antibody response was pronounced and uniform to GPIC; it was less marked to 6BC and LB-1, with fewer cross-reactions than seen in tests for cell-mediated immunity.

Cell-mediated immune responses to chlamydial antigens in guinea pigs injected with inactivated chlamydiae.

Cell-mediated immunity (CMI) to chlamydial antigens was readily induced in guinea pigs by a single injection of Betaprone-inactivated chlamydiae in complete Freund adjuvant. The CMI was measured in vivo by delayed hypersensitivity skin tests, and in vitro by inhibition of migration of peritoneal exudate cells and by proliferation of lymph node lymphocytes. There was an overall correlation between in vivo and in vitro responses. Of the in vitro assays, migration inhibition reflected the state of sensitization, as judged by skin tests, more uniformly than lymphocyte stimulation. Extensive inter- and intra-species cross-reactivity was noted between LB-1, a strain of C. trachomatis, and three strains of C. psittaci, 6BC, GPIC, and 562F. Cross-reactivity between LB-1 and 6BC was one-way only, by all three parameters: LB-1 elicited strong cross-reactions in 6BC-immunized animals but not vice versa. Antichlamydial antibodies could not be demonstrated in any of the animals by microimmunofluorescence.

Modified fluorometric determination of vitamin A in milk.

A modified fluorometric procedure for determination of vitamin A in milk was developed to provide rapid analysis of large numbers to samples.. Saponification and a single extraction in a reaction vessel without transfer provided simplicity and standardization. Time and temperature of saponification and time of extraction were studied. Fifteen replicates of four different milks gave standard deviations of .71, .82, 1.26, and .71 on samples with 29, 26, 38, and 26 mug retinol per 100 ml. Recovery of added vitamin A in six amounts in two experiments gave ranges of recovery of 96.0 to104.0 and 95.2 to 105.3% with average recoveries of 100.0 and 99.9%.

In vitro correlates of delayed hypersensitivity in man: ambiguity of polymorphonuclear neutrophils as indicator cells in leukocyte migration test.

Delayed cutaneous hypersensitivity (DCH) of 12 normal adult subjects to purified protein derivative (PPD) of Mycobacterium tuberculosis, streptococcal streptokinase-streptodornase (SK-SD), and Candida albicans Dermatophytin O (DO) was assayed in vivo by skin testing and compared with such in vitro correlates of cellular immunity as lymphocyte transformation (LT) and inhibition of leukocyte migration (ILM) from microcapillary tubes or in agarose gel. LT was shown to be the best in vitro correlate of specific lymphocyte sensitization with all antigens. In the ILM assays, PPD showed good correlation with in vivo DCH and in vitro LT; SK-SD showed partial correlation; DO showed no correlation, not being active in any of the ILM tests. Cell distribution and morphology of stained migration patterns, ILM tests performed on separated populations of lymphocytes and polymorphonuclear leukocytes (PMN), as well as the ability of test antigens to stimulate PMN cells to reduce nitroblue-tetrazolium dye, indicated that in ILM tests mononuclear cells were not inhibited in their migration, whereas migration of PMN cells appeared to depend on their direct reaction with the test antigens.