Herndon Douglas

Treatment of Gram-Negative Bacteremia and Shock with Human Antiserum to a Mutant Escherichia coli

Abstract In an effort to decrease deaths from gram-negative bacteremia and endotoxin shock, we treated bacteremic patients with human antiserum to endotoxin (lipopolysaccharide) core. Antiserum was prepared by vaccinating healthy men with heat-killed Escherichia coli J5; this mutant lacks lipopolysaccharide oligosaccharide side chains, so that the core, which is nearly identical to that of most other gram-negative bacteria, is exposed for antibody formation. In a randomized controlled trial, patients were given either J5 antiserum or preimmune control serum intravenously, near the onset of illness. The number of deaths in the bacteremic patients was 42 of 109 (39 per cent) in controls and 23 of 103 (22 per cent) in recipients of J5 antiserum (P = 0.011). In those with profound shock, mortality was 30 of 39 (77 per cent) in controls and 18 of 41 (44 per cent) in recipients of J5 antiserum (P = 0.003). We conclude that human antiserum to the lipopolysaccharide core can substantially reduce deaths from gram-...

Selective Protection against Conidia by Mononuclear and against Mycelia by Polymorphonuclear Phagocytes in Resistance to Aspergillus: OBSERVATIONS ON THESE TWO LINES OF DEFENSE IN VIVO AND IN VITRO WITH HUMAN AND MOUSE PHAGOCYTES

By comparing natural immunity to Aspergillus fumigatus (AF) in vivo with the action of human or mouse phagocytes against AF in vitro, we delineated two sequential lines of defense against AF. The first line of defense was formed by macrophages and directed against spores. Macrophages prevented germination and killed spores in vitro and rapidly eradicated conidia in vivo, even in neutropenic and athymic mice. The second was the neutrophilic granulocyte (PMN), which protected against the hyphal form of AF. Human and mouse PMN killed mycelia in vitro. Normal, but not neutropenic mice, stopped hyphal growth, and eradicated mycelia. Either line of defense acting alone protected mice from high challenge doses. Natural immunity collapsed only when both the reticuloendothelial system and PMN were impaired. These findings are in keeping with the clinical observation that high doses of cortisone and neutropenia are the main risk factors for invasive aspergillosis. Cortisone inhibited the conidiacidal activity of mouse macrophages in vivo and of human or mouse mononuclear phagocytes in vitro. Cortisone damaged this first line of defense directly and not through the influence of T lymphocytes or other systems modifying macrophage function as shown in athymic mice and in vitro. In addition, daily high doses of cortisone in mice reduced the mobilization of PMN so that the second line of defense was also impaired. Thus, cortisone can break down natural resistance on its own. Myelosuppression rendered mice susceptible only when the first line of defense was overpowered by high challenge doses, by activated spores that cannot be killed by macrophages, or by cortisone suppression of the conidiacidal activity of macrophages. The host, thus, can call upon two independent phagocytic cell lines that form graded defense systems against aspergillus. These lines of defense function in the absence of a specific immune response, which seems superfluous in the control and elimination of this fungus.

In vitro susceptibility of fungi to killing by neutrophil granulocytes discriminates between primary pathogenicity and opportunism.

Pathogenic fungi, according to their propensity to cause infection of apparently normal individuals, can be grouped into either primary pathogens (e.g., Coccidioides, Histoplasma, Paracoccidioides, Blastomyces, and Sporothrix) or opportunists (e.g., Candida, Mucoraceae, Aspergillus spp., Petriellidium, and Trichosporon). There is, however, no unifying concept explaining the difference between the virulence of the two fungal categories. Previously we have speculated that neutrophils are the common denominator of the high natural resistance to opportunistic fungi. Accordingly, we then compared the susceptibility to killing by neutrophil granulocytes of Histoplasma, Blastomyces, Paracoccidioides, and Sporothrix with that of 14 opportunistic fungi. We found the four virulent dimorphic yeasts, in contrast to opportunistic fungi, to be resistant to killing by neutrophils. Virulent dimorphic yeasts were ingested by neutrophils, and triggered a respiratory burst comparably to opportunists but were less susceptible to hydrogen peroxide, suggesting that differences in the susceptibility to microbicidal products of leukocytes may explain the difference in virulence.

Induction of immunity against lethal Haemophilus influenzae type b infection by Escherichia coli core lipopolysaccharide.

Efforts to prevent Haemophilus influenzae type b (HIB) infections in infancy have been hampered by the low immunogenicity of capsular polysaccharide vaccines in children younger than 18 mos. In searching for alternate immunogens, we have studied the protective potential of polysaccharide-poor, lipid-rich endotoxin (LPS) core in experimental HIB infections. Because all gram-negative bacteria have similar LPS core structures, we were able to use as vaccine the J5 mutant of Escherichia coli 0111, the LPS of which consists only of core components, and thus to avoid problems in interpretation arising from vaccine contamination with non-LPS HIB immunogens. Mice were given graded inocula of HIB and developed lethal infection analogous to human HIB disease when virulence was enhanced with mucin and hemoglobin. After active immunization with heat-killed E. coli J5, 40/50 (80%) of infected mice survived, compared with 14/50 (28%) of saline-immunized controls (P less than 0.005). Passive immunization with rabbit antiserum against E. coli J5 prevented lethal HIB infection when administered 24 or 72 h before or 3 h after infection. This protection was abolished by adsorption of antiserum with purified J5 LPS, with survival reduced from 14/24 to 0/24 (P less than 0.005). Furthermore, rabbit antiserum to purified J5 LPS gave just as potent protection against death as antiserum to whole J5 cells. These studies demonstrate that immunity to core LPS confers protection against experimental murine HIB infection and provide the framework for a new approach to prevention of human disease from HIB.

Fibrin-enmeshed Tobramycin Liposomes: Single Application Topical Therapy of Pseudomonas Keratitis

Treatment of bacterial keratitis requires frequent application of topical antibiotics. We studied the efficacy of a single topical administration of tobramycin incorporated in large multivesicular liposomes and enmeshed in a fibrin sealant on rabbit corneas infected with Pseudomonas aeruginosa. One cornea each of 25 New Zealand albino rabbits was infected with P. aeruginosa. Twenty-four hours later, the animals were randomly divided into five groups of five. Group A received single hourly drops (50 μl) of fortified tobramycin (14.5 mg/ml, total of 17.4 mg). Group B received a single topical application of 3.5 mg tobramycin, in 0.1 ml multivesicular liposomes, enmeshed in a fibrin sealant with an overlaying bandage contact lens. Group C was treated in the same manner as group B without the addition of fibrin sealant. Groups D and E served as nondrug-treated controls, with group D receiving topical fibrin-enmeshed liposomes devoid of tobramycin and group E receiving hourly topical balanced salt solution (BSS) drops. All animals were killed 24 h after initiation of therapy. Significantly fewer colonies of Pseudomonas were present in corneas of all three treated groups, as compared with the two nondrug-treated control groups (p < 0.02). There were significantly fewer colonies of Pseudomonas in groups A and B as compared with group C (p < 0.02). No significant difference was noted between a single administration of topical fibrinenmeshed tobramycin-encapsulated liposomes (group B) and 24 doses of hourly fortified topical tobramycin (group A, p > 0.05). Tobramycin-encapsulated megaliposomes may serve as a useful adjunct in treatment of Pseudomonas keratitis.

Human Antiserum for Prevention of the Local Shwartzman Reaction and Death from Bacterial Lipopolysaccharides

Bacterial lipopolysaccharides from dead bacteria have been blamed for the continuing high mortality from gram-negative infections despite antibiotic treatment. Because animal antiserum against these lipopolysaccharides has been shown to protect against several of the effects of endotoxin, we undertook the development of antiserum in human subjects. 21 men were immunized with a single injection of Salmonclla typhimurium or Escherichia coli 0:111 heat-killed cells and immune serum was collected at 2 wk. Preimmune serum was obtained as a control in all animal experiments. 1 ml antiserum given intravenously protected mice against a lethal intravenous dose of homologous endotoxin (P < 0.005 for both antisera). E. coli antiserum reduced the incidence of positive local Shwartzman reactions with E. coli endotoxin from 100 to 38%; S. typhimurium antiserum reduced the incidence from 92 to 35%. (P < 0.0005 for both antisera). There was no protection against heterologous endotoxin in either animal model. These experiments demonstrate for the first time that human antiserum confers exceedingly potent passive immunity to the effects of endotoxin.

Lethal Haemophilus influenzae type b infection in mice

SummaryPrevious animal models of invasiveHaemophilus influenzae type b (HITB) infection are characterized by a low mortality rate. We produced a highly lethal infection in CF1 mice using mouse passage, mucin, and hemoglobin to enhance infectivity. Infection by the intraperitoneal route was followed by progressive peritonitis and bacteremia with subsequent HITB infection of the brain and meninges, and death. Death occurred between eight and 72 hours after infection and was associated with 106 to 109 HITB per ml of blood and with 102 to 105 HITB per g of brain. Mucin-hemoglobin did not augment HITB growth, but impaired macrophage adherence to glassin vitro, without decreasing cellular viability.In vivo, mucin-hemoglobin decreased the rate of disappearance of51Cr-labelled HITB from the blood by impairment of hepatic clearance. This technically simple and inexpensive model is useful for the study of HITB infections in which bacterial multiplication, invasion and host lethality are desired features.ZusammenfassungHerkömmliche Tiermodelle für die invasiveHaemophilus influenzae Typ b-(HITB-)Infektion haben nur eine geringe Letalität. Durch erhöhte Infektiosität des Erregers über Mäuse-Passage sowie mit Mucin und Hämoglobin konnten wir bei CF1-Mäusen eine Infektion mit hoher Letalität hervorrufen. Nach intraperitonealer Infektion entwickelte sich eine progressiv verlaufende Peritonitis und Bakteriämie, der eine HITB-Infektion des Gehirns und der Meningen mit letalem Ausgang folgte. Der Tod trat zwischen acht und 72 Stunden nach der Infektion ein, die Keimzahlen im Blut lagen zwischen 106 und 109 HITB pro ml, im Gehirn fanden sich 102 bis 105 HITB pro g. Mucin-Hämoglobin führte nicht zu einer Steigerung des HITB-Wachstums, doch hemmte esin vitro die Makrophagenadhäsion an Glas, ohne die Überlebenszeit der Zellen zu verändern.In vivo führte Mucin-Hämoglobin durch Störung der hepatischen Clearance zu einer Verminderung der Eliminationsrate von51Cr-markierten HITB aus dem Blut. Dieses technisch einfache und wenig aufwendige Model eignet sich zum Studium von Infektionen durch HITB, die die Charakteristika Bakterienvermehrung, Invasion und tödlicher Ausgang haben sollen.

A new method for purification of Giardia lamblia cysts

One of the major problems in studying Giardia cysts is separating this form of the parasite from faecal debris and bacteria. We approached this problem by passing filtered stool suspensions over a column of Sephadex G-50 followed by low speed centrifugations. The Sephadex retained most of the faecal debris and the centrifugation reduced the bacterial flora more than 10(5)-fold to less than 3 X 10(4)/ml. Cyst recovery was 61% (+/- 28%). This new method of separating G. lamblia from faecal material is simple, rapid, and effective.