Robert C. Fader

Elevated cAMP in intestinal epithelial cells during experimental cholera and salmonellosis

Cholera and salmonellosis are two diarrheal diseases in which intestinal tissue cyclic adenosine monophosphate (cAMP) concentrations are elevated. Investigations of each experimental disease were initiated to identify the specific intestinal cells containing the elevated cAMP. Epithelial cells were eluted from the mucosa of infected and control intestinal loops of adult rabbits, after which the cAMP content of the epithelial cell fractions and the lamina propria cells was extracted and assayed. The identity of the epithelial cells (in the villus tip-to-crypt cell gradient) was monitored by measuring their intracellular alkaline phosphatase activity, while scanning electron microscopy was used to visualize the effects of infection and cell elution techniques. Clearly, in both experimental cholera and salmonellosis, elevated cAMP levels were associated with crypt epithelial cells. Villus tip epithelial cells from either infection tended to contain less cAMP than those of noninfected control tissue. In Salmonella-infected loops, it was apparent that cAMP was also elevated in lamina propria cell fractions. Lamina propria cells from V. cholerae-infected intestinal loops contained only basal levels of cAMP. In vitro exposure of isolated intestinal cells from normal rabbit intestine to a cell-free lysate of Salmonella resulted in elevation of cAMP in the epithelial cells and lamina propria cells. We conclude that in experimental cholera and salmonellosis, significant elevation of the cAMP levels occurred in intestinal crypt cells, consistent with an enterotoxin-mediated mechanism. In Salmonella-infected loops, it was unclear if the increased concentration of cAMP in lamina propria cells was generated by enterotoxin released from the invasive salmonellae or by prostaglandins formed during the inflammatory response to the bacteria, or by both mechanisms.

Klebsiella Pneumoniae-Induced Experimental Pyelitis: The Effect of Filiation on Infectivity

The effect of bacterial piliation on the infectivity of Klebsiella pneumoniae in the renal pelvis was examined by means of piliated and nonpiliated variants derived from a single parent strain. Piliated-phase variants were significantly more infective as determined by viable counts of bacteria isolated from the kidneys at the time of sacrifice. In addition, scanning electron microscope was used to examine the renal pelvis for evidence of infection. Kidneys infected with piliated-phase organisms exhibited greater tissue damage -- both in the magnitude of tissue alterations observed and in the number of kidneys affected. Thus, it is concluded that piliation contributes to the ability of K. pneumoniae to infect the renal pelvis following reflux from the bladder.

Exaggerated cardiopulmonary response after bacteremia in sheep with week-old thermal injury

Objective.To determine if thermal injury impairs pulmonary intravascular clearance of bacteria and therefore leads to exaggerated cardiopulmonary dysfunction in sheep, since endotoxin infusion has been previously shown to induce more severe pulmonary injury after thermal injury. Design.Prospective, unblinded, randomized, controlled trial. Setting.Laboratory at a large university medical center. Interventions.Chronically instrumented, anesthetized sheep received a 40% total body surface area, third-degree thermal injury. Live Pseudomonas aeruginosa (107P. aeruginosa/min for 1 hr; n = 6) were infused 7 to 10 days after thermal injury. Similarly prepared noninjured sheep received the same pseudomonas infusion (n = 7) or saline (n = 7). Measurements and Main Results.Bacterial clearance, which measures phagocytosis by the pulmonary intravascular macrophages, was equally efficient in intact sheep and sheep with thermal injury. Pulmonary hypertension persisted for 18 hrs after thermal injury, compared with 8 hrs in noninjured sheep. Lung lymph flow significantly increased from 6 to 8 hrs in only the thermal injury group. Both bacteremic groups developed a hyperdynamic circulation from 6 to 8 hrs, but cardiac index was 1 to 1.5 L/min/m2 higher in thermally injured sheep. Total peripheral resistance index decreased significantly from 6 to 24 hrs in thermally injured sheep and from 6 to 12 hrs in intact bacteremic sheep. Mean arterial pressure of thermally injured sheep was increased at baseline and for the first 6 hrs compared with noninjured animals. Mean arterial pressure decreased from 6 to 24 hrs in sheep with thermal injury but did not change in intact bacteremic sheep. Conclusions.Bacterial clearance was not impaired by preceding thermal injury in sheep. Bacteremia in the presence of a preexisting thermal injury led to more persistent pulmonary hypertension and an exaggerated hyperdynamic circulation. (Crit Care Med 1993; 21:88–893)

Expression of hemagglutination is dependent on environmental factors and bacterial concentration ofEscherichia coli andKlebsiella pneumoniae

Hemagglutination of human type A and guinea pig erythrocytes showed that both mannose-sensitive and mannose-resistant adhesive mechanisms (pili) were detectable in broth cultures ofEscherichia coli while only a mannose-sensitive mechanism (type 1 pili) was noted withKlebsiella pneumoniae. The degree of hemagglutination was shown to be related to differences in erythrocytes, bacterial concentration, and growth media constituents.Escherichia coli grown in human urine produced both types of hemagglutinins, whileK. pneumoniae cultured in urine continued to express only a mannose-sensitive hemagglutinin.

Inhibition of bacterial adherence to rat bladder epithelial cells by human immune serum globulin

The ability of commerical human immune serum globulin (HISG) to inhibit the adherence of urinary tract infection isolates to rat bladder epithelial cells was investigated utilizing an in vitro adherence system. Significant decreases in adherence were noted when strains ofEscherichia coli, Klebsiella pneumoniae, Proteus mirabilis, andEnterobacter cloacae were tested against five HISG preparations. An enzyme-linked immunosorbent assay indicated that all five HISG preparations also contained antibodies against type-1 pili isolated fromKlebsiella pneumoniae. The presence of antibodies directed against a bacterial adhesin and the effectiveness of HISG in inhibiting the attachment of a wide range of urinary pathogens to bladder cells suggest that HISG may have practical therapeutic values in the prophylaxis of diseases where bacterial adherence is a prerequisite for the initiation of infection.

Effect of Preexisting Inhalation Injury on Response to Bacteremia in Sheep

Inhalation injury increases the likelihood of sepsis. We tested the hypothesis that preexisting inhalation injury would diminish bacterial clearance across the pulmonary vasculature and induce greater hemodynamic response. Live Pseudomonas aeruginosa were infused centrally for one hour in three groups of awake sheep. Inh + Ps (n = 10), with a seven to 10-day-old inhalation injury; Ps-LOW (n = 8) both received 10(7) Ps/min; and Ps-HI (n = 9) received 5 x 10(7) Ps/min. for one hour. Pulmonary hypertension was more severe in Ps-HI and Inh + Ps. A hyperdynamic response with high cardiac index and low mean arterial pressure developed in Inh + Ps and Ps-HI from six to 18 hr, while Ps-LOW, only the cardiac index was elevated at six to eight hr. The Inh + Ps and Ps-LOW groups had equivalent pulmonary artery and aortic bacterial levels, while Ps-HI had levels approximately five times higher. All groups removed bacteria efficiently in the lungs. However, preexistent inhalation injury exaggerated the pulmonary and systemic hemodynamic response.

Systemic candidiasis after thermal injury

The effect of thermal injury on the host response to systemic infections withCandida albicans was studied by use of a 20% total body surface, full-thickness scald burn in inbred BALB/c mice. The lethal dose of intravenously injectedC. albicans required to kill 50% of the population (LD50) of control, sham-burned mice was 4.7×105 organisms, whereas the LD50 of burned mice was 300 organisms (p<0.001). Mice injected intraperitoneally with 2×107C. albicans were assayed at two, four, seven, and ten days after burn for the presence of yeasts in the kidneys, skin, or burn wounds. At each time period examined, fewer yeasts were recovered from the kidneys of sham-burned mice than from burned mice. In addition, only one (5%) of 20 sham-burned mice had organisms in the skin at the time of sacrifice, whereas seven (37%) of 19 burned mice had organisms isolated from the burn wound (p<0.05). Wound histology demonstrated that the organisms were localized in the subeschar area and not colonizing the wound surface. This study describes the enhanced susceptibility of burned mice to systemic candidiasis and shows that a systemic infection withCandida can lead to organisms contaminating the wound.

BACTERIAL AGENTS STIMULATE THE RELEASE OF SECRETORY IGA FROM HUMAN MILK LEUKOCYTES

Recently we reported that the secretion of secretory IgA (SIgA) from human milk leukocytes was stimulated by phagocytosis (J. Infect. Immun. 34: 498, 1981) and certain surface membrane stimuli including N-formyl-L-methionyl-L-phenylalanine (J. Immunol. In Press, 1984). Since that synthetic peptide is similar to ones secreted by certain strains of enteric bacteria, we questioned whether other bacterial products also trigger the release of that immunoglobulin from phagocytes in human milk. Supernatent fluids from cultures of Escherichia coli 07KL or Klebsiella pneumoniae obtained by passage through 0.22μ filters lead to the release of 40-50% of the SIgA found in unfractionated human colostral leukocytes (p<0.01). A similar degree and temporal pattern of SIgA release occurred when those leukocytes were exposed to heat-treated filtrates from those bacterial cultures, purified lipopolysaccharide from E. coli or type 1 pili isolated from K. pneumoniae.Thus, the secretion of SIgA by human milk leukocytes is initiated not only by phagocytosis of intact microorganisms, but also by important components of certain enteric bacterial pathogens. Furthermore, these findings support the hypothesis that SIgA released from those leukocytes plays a role in protecting the breast-fed infant from bacterial infections of the gastrointestinal tract.