Prolonged antigen half-life in the lymphoid follicles of specifically immunized mice

The kinetics of clearance of ¹²⁵I from the popliteal lymph nodes and feet of human serum albumin (HSA)-immunized mice was studied following the injection of [¹²⁵I]-HSA into the hind footpads. Antigen was cleared from both locations rapidly for the first few days. The antigen half-life (T½) during this period was only a matter of hours. By the end of the first week, however, the rate of clearance in both sites had changed markedly. The antigen T½ in the node between the first and sixth week was 8.1 weeks (95% confidence interval between 5.1 and 20 weeks) and the antigen T½ in the foot was 6.1 weeks (95% confidence interval between 3.7 and 16.6 weeks). There was, however, about twenty times more radioactivity in the feet than in the popliteal nodes. Autoradiography of popliteal lymph nodes revealed that initially antigen was trapped in the medulla, subcapsular sinus, superficial cortex and around lymphoid follicles. During the first few days antigen was cleared from all sites except the follicles. The radioactivity initially trapped in the medulla, subcapsular sinus, and superficial cortex appeared to have been associated with macrophages. Studies with peritoneal macrophages indicated an antigen T½ in these cells of 2 h (95% confidence interval between 1.5 and 3 h). The initial rapid clearance of antigen trapped and catabolized by macrophages and the long-term retention of antigen in the follicles is probably attributable to trapping and retention by follicular dendritic cells. The large pool of antigen trapped in the foot did not appear to serve as a depot to replace antigen degraded in the node, since amputation of the foot did not alter the level of antigen retained in the node. The long antigen T½ in the lymph node follicles indicates that antigen is available in the lymph node to play a role in the maintenance and regulation of immune responses for many months or even years.     

In Vivo Behavior of Fibrinogen Fragment D in Experimental Renal, Hepatic and Reticuloendothelial Dysfunction

Fibrinogen Fg-D, obtained by plasmin-induced cleavage of fibrinogen, was separated and purified by ion exchange chromatography. The in vivo behavior was studied by administering 2 mg of ¹²⁵I-labeled Fg-D intravenously into each of 3 normal, 3 partially hepatectomized, 3 reticuloendothelial system (RES) blockaded, 4 nephrectomized and 2 ureter ligated rabbits. The plasma clearance in normal rabbits showed two components: 66.0 ± 6.0% was cleared with a t½ of 0.9 ± 0.2 hours and 32.3 ± 5.3% cleared with a t½ of 3.6 ± 0.3 hours. In both the partially hepatectomized and RES-blockaded groups, the clearance patterns were similar to those observed in the normal animals. In contrast, in the nephrectomized group, while the t½ of the first component was similar to that in the normal group, the second component had a longer t½ (6.0 ± 1.0 hours) than the other groups. In the animals with both ureters occluded, the t½'s were similar to the normal animals. Measurements of urinary radioactivity suggested that complete in vivo catabolism had occurred. In vivo subfragments of Fg-D could not be detected in the plasma. Neither Fg-D nor subfragments were found in the urine. These results indicate that Fg-D is rapidly cleared from the plasma, that in vivo subfragmentation does not occur, and that the kidneys are important in the catabolism of a portion of Fg-D.     

The Fate of [131I]Labelled Antigens in the Skin of Normal Guinea-Pigs and those with Delayed-Type Hypersensitivity

A comparison was made of the retention of three antigens PPD (protein purified derivative of tuberculin), HSA (human serum albumin) and HGG (human γ globulin) labelled with ¹³¹I in the skin of normal guinea-pigs. It was found that PPD was eliminated more rapidly than HSA or HGG in the first 24 hours after skin test. In contrast PPD was found to be more readily adsorbed to collagen and cellular tissues in vitro.

The retention of these antigens at 24 hours in normal and sensitized guinea-pigs was also compared. It was found that PPD was retained in the tuberculin lesion as compared with normal skin, whereas HSA was eliminated more rapidly from a delayed-type hypersensitivity lesion to HSA when compared with normal skin. It was concluded that retention of PPD was probably not specific to delayed-type hypersensitivity but was related to the difference in character and severity of the inflammatory process, which itself was related to the chemical nature of the antigen involved.

     

Mouse peritoneal cells: their ability to elicit or produce antibody after exposure to antigen

The functional activities of peritoneal cells in exudates of normal mice were studied.

Antigen-containing peritoneal cells were tested for their ability to induce antibody by transfer into normal syngeneic recipients. Collection of peritoneal cells at varying time intervals following intraperitoneal injection of formalin-killed pneumococci or capsular polysaccharide, resulted in a decrease with time in the ability of these cells to induce antibody in the recipient mice. This finding could be ascribed to changes in the cell population in the peritoneum as a result of antigen administration rather than to loss of immunogenic material from these cells. Evidence for this comes from experiments in which peritoneal cells collected ½-hour after antigen administration were maintained in vitro for up to 24 hours. The immunogenicity of carbohydrate antigen in these cells persists for at least 24 hours. Furthermore our morphological observations showed a drop in macrophages following antigen administration.

Our findings indicate that peritoneal exudates also contain immunocompetent cells. For this study we used recipient mice irradiated with 650–800 r, which completely suppressed their antibody response. Peritoneal cells collected 2 hours after injection of formalin-killed pneumococci led to serum antibody in the irradiated recipients 7 days later. By partially fractionating peritoneal cells on glass surfaces, it could be shown that the cells not adhering to glass (lymphocytes and medium sized cells) were mainly responsible for this antibody response. Cells adhering to glass which contained most of the antigen-containing large macrophages and also medium sized cells, were much less effective in inducing antibody. In addition, peritoneal cells induced with thioglycollate medium consisting mainly of large macrophages and very few lymphocytes, actively induced antibody in normal recipients but failed to restore antibody production in the highly irradiated mice. The transfer of antibody formation to irradiated mice thus is due to immunocompetent cells in normal peritoneal exudates.

     

Immunological responsiveness following the continuous circulation of soluble antigen—antibody complexes

Following the continuous circulation of soluble bovine serum albumin—anti-bovine serum albumin complexes, adult rabbits rapidly lose the ability to synthesize specific antibodies to bovine serum albumin (BSA). This tolerant state appears to be similar to that produced in adult rabbits by large doses of antigen.

(1) The animals showed delayed elimination of injected [¹²⁵I]BSA.

(2) Immunization with human serum albumin (HSA) enhanced the elimination of intravenously injected [¹²⁵I]BSA.

(3) BSA failed to stimulate the in vitro incorporation of [¹⁴C]thymidine by spleen cell suspensions from these tolerant animals.

(4) Although some animals made a small amount of anti-BSA antibodies in vivo there was no response in vitro to the same antigen.

     

The localization of non-microbial antigens in the draining lymph nodes of tolerant, normal and primed rabbits

Soluble haemocyanin (HCy) or human serum albumin (HSA) labelled with ¹²⁵I at 8 and 0·7 μc/μg, respectively, were injected into the footpads of rabbits in doses just sufficient to elicit a primary response in normal animals, and the distribution of radioactivity in the popliteal lymph nodes between 6 hours and 21 days later was studied by autoradiography. The recipient rabbits had either been primed by a single prior injection of unlabelled antigen, or made putatively tolerant by repeated neonatal administration of antigen, or were previously untreated. Localization of antigen in germinal centres, in a typical dendritic pattern, was marked in the primed animals throughout the period of observation; in those tolerant animals which had no detectable serum antibody initially and made no detectable antibody response such localization was not seen at any time; in the animals that had no previous contact with antigen there was no localization in germinal centres until about the time when antibody became detectable in the serum. Localization of radioactivity in medullary sinus macrophages did not differ significantly between the three groups.

It is concluded that localization of these soluble antigens on the dendritic web in lymphoid follicles occurs as a consequence of the presence of circulating antibody. Uptake of the antigens by medullary macrophages, however, can occur in the absence of antibody. Although the degree of labelling of medullary macrophages was not evidently affected by the presence of antibody in these experiments, it is emphasized that the antibody levels, even in the primed animals, were low, and that this finding is unlikely to apply when the amount of antibody present is relatively much greater than the amount of antigen injected.

     

Characteristics of the 19S and 7S response to bacteriophage ΦX174 in the fowl

After intravenous injection of 10¹⁰ plaque forming particles of ØX174 bacteriophage into White Rock fowls, immune elimination began at 30 hours and viable phage was cleared from the circulation by 50–52 hours, the approximate time at which detectable antibody appeared. Little change was noted in the serum neutralizing activity in the interval from 44 to 217 days after injection, at which time the birds were reinjected with ØX174.

Sephadex Peak I (19S globulin) accounted for most of the early activity of the primary response. By Day 9 most of the activity had shifted to Peak II (7S globulin). In the secondary response, the shift had occurred by Day 4. Both 19S and 7S globulin fractions showed an increase in activity when compared to the same days of the primary response, but the 7S increase was proportionately greater.

All sera and serum fractions (whether 19S or 7S) were sensitive to reduction with 2-mercaptoethanol, but became, less sensitive during the course of immunization.

With regard to the production of 19S antibody, White Rock fowls showed a different response to the particulate antigen ØX174 compared with the response to injection of the soluble antigen, bovine serum albumin. There is evidence of immunological memory in the 19S response to ØX174.

     

The action of soluble antigen—antibody complexes in perfused guinea-pig lung

When soluble antigen—antibody complexes, prepared in antigen excess, were injected into the perfused pulmonary artery of the unsensitized guinea-pig lung, they produced a bronchoconstrictor response which was associated with the appearance of histamine and a slow-reacting substance in the effluent. This activity was inhibited by the presence of normal rabbit serum or rabbit globulin; rabbit albumin and bovine γ-globulin produced no inhibition. Insoluble antigen—antibody complexes prepared at equivalence were at least 125 times less active than soluble complexes prepared in antigen excess. On comparing the activity of different solutions of soluble complex prepared in antigen excess ranging from 2½ to 160 times that required for equivalence, all were found to be equal. The properties of soluble antigen—antibody complexes are consistent with the possibility that circulating antibody may participate in in vivo anaphylaxis, if reached by an amount of antigen greater than that required for equivalence.     

The Distribution of Prostaglandins in Afferent and Efferent Lymph From Inflammatory Sites

Arachidonic acid labeled with ¹⁴C was injected directly into lymph nodes that had been stimulated at various times with Escherichia coli. The efferent lymph was collected, and labeled catabolites were extracted and analyzed chromatographically. The pimary conversion product recovered was Prostaglandin E₂ (PGE₂), with the lesser products thromboxane, prostacyclin and prostaglandin F_2α (PGF_2α) also detected. When the efferent lymph was analyzed by radioimmunoassay after subcutaneous injectino of E coli into the hock, PGE and PGF levels rapidly increased, reached the highest levels in the first 10 hours, and then returned to normal by 24 hours. When the afferent lymph plasma draining inflammatory sites was compared directly with efferent lymph, PGF levels were similar, but the PGE level was always several times higher in the afferent lymph. To examine the catabolism of PG, either ^3,H-PGF_2ά of ³H-PGE₂ was injected into the node, and the efferent lymph plasma was analyzed. No conversion of PGF_2α to other products was found. In contrast, catabolic products of PGE₂ were detected. With the use of equilibrium dialysis techniques, the binding of PGE₂ and PGF_2α to proteins in lymph and to bovine serum albumin (BSA), human serum albumin (HSA), and BSA stripped of its fatty acids was established. The binding to lymph proteins correlated with the albumin concentrations in the lymph. This albumin binging probably facilitated the retention and transport of PG in the lymph. PG appears in the lymph at a time corresponding to the uptake and processing of antigen by the node and near the time when lymphokines are detected in lymph and could modulate several steps in the immune response. The PGE detectable in the lymph draining an inflammatory site may play a role in the modulation of blood flow.     

A sopB deletion mutation enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines

SopB is a virulence factor of Salmonella encoded by SPI-5. Salmonella sopB deletion mutants are impaired in their ability to cause local inflammatory responses and fluid secretion into the intestinal lumen and also can enhance the immunogenicity of a vectored antigen. In this study, we evaluated the effects on immunogenicity and the efficacy of a sopB deletion mutation on two Salmonella enterica serovar Typhimurium vaccine strains with different attenuating mutations expressing a highly antigenic α-helical region of the Streptococcus pneumoniae surface protein PspA from an Asd⁺-balanced lethal plasmid. After oral administration to mice, the two pairs of strains induced high levels of serum antibodies specific for PspA as well as to Salmonella antigens. The levels of antigen-specific serum immunoglobulin G (IgG) and mucosal IgA were higher in mice immunized with sopB mutants. Enzyme-linked immunospot assay results indicated that the spleen cells from mice immunized with a sopB mutant showed higher interleukin-4 and gamma interferon secretion levels than did the mice immunized with the isogenic sopB⁺ strain. The sopB mutants also induced higher numbers of CD4⁺ CD44^hi CD62L^hi and CD8⁺ CD44^hi CD62L^hi central memory T cells. Eight weeks after primary oral immunization, mice were challenged with 100 50% lethal doses of virulent S. pneumoniae WU2. Immunization with either of the sopB mutant strains led to increased levels of protection compared to that with the isogenic sopB⁺ parent. Together, these results demonstrate that the deletion of sopB leads to an overall enhancement of the immunogenicity and efficacy of recombinant attenuated Salmonella vaccine strains.     

The influence of adjuvants on the immunological response of the chicken: I. Effects on primary and secondary responses of various adjuvants in the primary stimulus

The effect of various adjuvant procedures on the antibody response (first 21 days) for human serum albumin (HSA) has been studied in the chicken. The lack of effect on the circulating antibody level contrasts with their action in some mammals.

The administration of depôt-type adjuvants failed to increase the peak circulating antibody levels (8–12 days after injection of antigen) by comparison with control birds. However, the circulating antibody level declined more slowly in birds given HSA in a water-in-oil emulsion than in birds given HSA in saline.

The administration of endotoxin and `surface active' adjuvants also failed to increase the peak circulating antibody levels over that of control birds. In three experiments there was significant depression of peak antibody levels in birds given endotoxin adjuvant in comparison to control birds.

The administration of HSA in Freund's complete adjuvants containing Mycobacterium tuberculosis or Mycobacterium avium did not result in elevation of peak antibody levels compared to those of control birds given HSA in saline or HSA in a water-in-oil emulsion.

Experiments to determine the effect of adjuvants from each of the main groups on the establishment of immunological memory were performed. Chickens were given adjuvant with the primary injection of HSA. A second injection of HSA without adjuvant was given 56 days later. None of the adjuvants used produced an increase in the peak antibody level attained during the secondary response compared to control birds.

     

Novel polymer-grafted starch microparticles for mucosal delivery of vaccines.

Recent studies have demonstrated that systemic and mucosal administration of soluble antigens in biodegradable microparticles can potentiate antigen-specific humoral and cellular immune responses. However, current microparticle formulations are not adequate for all vaccine antigens, necessitating the further development of microparticle carrier systems. In this study, we developed a novel microparticle fabrication technique in which human serum albumin (HSA) was entrapped in starch microparticles grafted with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS), a biocompatible silicone polymer. The immunogenicity of HSA was preserved during the microparticle fabrication process. Following intraperitoneal immunization of mice, TS-PDMS-grafted microparticles (MP) dramatically enhanced serum IgG responses compared with ungrafted MP and soluble HSA alone (P < 0.001). When delivered orally, both TS-PDMS-grafted and ungrafted microparticles elicited HSA-specific IgA responses in gut secretions, in contrast to orally administered soluble antigen. Indeed, TS-PDMS-grafted microparticles stimulated significantly stronger serum IgG (P < 0.005) and IgA (P < 0.001) responses compared with those elicited by ungrafted microparticles. These findings indicate that TS-PDMS-grafted starch microparticles have potential as systemic and mucosal vaccine delivery vehicles.     

Antigen in tissues: IV. The effect of antibody on the retention and localization of antigen in rat lymph nodes*

A study has been made of the role of specific antibody in causing increased retention and specific localization of a weak antigen, human serum albumin (HSA), in the popliteal and aortic lymph nodes of rats. The antigen was labelled with ¹²⁵I prior to mixing with antibody. HSA mixed with excess homologous antibody was trapped to the greatest extent in these nodes after footpad injection of the antigen. Injection of HSA with antibody caused increased uptake of HSA into the medulla but retention was poor as autoradiographs showed the area to be essentially free of antigen 4–5 days after injection. By contrast, antigen injected with antibody localized strongly in lymphoid follicles and persisted at this site. Both IgM and IgG antibody were found to cause follicular localization of HSA in rats. Heterologous, isologous and homologous antibody also caused follicular localization of the antigen. Purified homologous γ-globulin was found to localize in the follicles. A moderate increase in the net negative charge of the γ-globulin by acetylation did not appreciably affect the ability of the globulin to localize in the follicles. Detectable formation of antibody did not occur in the rats after injection of antigen—antibody complexes, owing possibly to the inhibitory effect of free antibody on the primary response.     

Mucosal penetration of antigen in the presence or absence of serum-derived antibody: An in vitro study of rabbit oral and intestinal mucosa

Rabbit mucous membranes were mounted in diffusion chambers within 30 min after excision. The epithelial side was exposed to cell culture medium containing `cold' and ¹²⁵I-labelled human albumin. The basal side was exposed to normal rabbit serum (control chambers) or to rabbit anti-serum against human albumin. The chambers were incubated in a humidified atmosphere of CO₂ in air for 2–3 h at 37°. The radioactive material recovered on the basal side of the sublingual control membranes sedimented virtually like native albumin on ultracentrifugation. The amount of radioactive material recovered after penetration through anti-serum-exposed sublingual mucosa was reduced by 50–80% and showed a very heterogeneous sedimentation pattern including aggregates, presumably immune complexes, as well as a considerable amount of degradation products.

In a second series of experiments the concurrent penetration of human albumin and transferrin through the sublingual mucosa of rabbits immunized parenterally with albumin was compared with that occurring through control membranes. With reference to immunochemical quantification, scintillation counting was found to overestimate the penetration of intact albumin considerably, and jeopardized evaluation of the influence of serum-derived antibody. Radial immunodiffusion showed that in controls the basal antigen concentration, expressed in percentage of the oral (30 mg/ml for both molecules), was after 2 h 0.0032±0.0023 for albumin and 0.0016±0013 for transferrin. Penetration of immunoreactive albumin through mucosa from immunized animals was clearly reduced (0.0007±0.0003), whereas there was a significant tendency toward increased penetration of transferrin (0.0035±0.0023). These results suggest that antibody within the mucosa retards the penetration of intact homologous antigen, while immune reactions may enhance the penetration of unrelated macromolecules.

In similar experiments with colon mucosa penetration was 50–100 times increased, but the membranes did not discriminate between albumin and transferrin and there was no effect of immunization. Histological and immunohistochemical studies of the latter membranes indicated marked defects in cell viability after 2 h in vitro.

     

Induction of oral tolerance with effects on numbers of IgE-carrying mast cells and on bystander suppression in young rats

The presence of IgE⁺ mast cells in the small intestine, bystander suppression of DTH and antibody responses to human serum albumin (HSA) were studied in young rats, made tolerant to ovalbumin (OA) by feeding an OA-containing diet for 1–4 weeks starting from weaning, and in sensitized control rats. One week after finishing the OA diet, both groups of rats were immunized with a mixture of OA and HSA in Freund’s complete adjuvant (FCA) at one site on the back. The animals were then colonized for 5 days with a genetically manipulated Escherichia coli producing OA. Immunohistochemical staining of the small intestine of the rats fed the OA diet for 4 weeks showed significantly fewer IgE⁺ mast cells in the lamina propria, a lower level of MHC class antigen was found in the epithelial cells and in the lamina propria, and the villus crypt depth was also significantly less in tolerant compared with sensitized rats (P<0.003, 0.007, 0.003, respectively). Sensitized rats showed a mild diarrhoea during the colonization in contrast to tolerant rats. All rats fed OA showed a significantly reduced IgE anti-OA antibody and DTH response to OA before colonization compared with the sensitized rats. Bystander suppression of IgG and IgE anti-HSA antibody responses was also seen, but only in the rats fed OA for either 1 or 4 weeks. Rats fed the OA-containing diet for 1, 3, or 4 weeks showed bystander suppression of the DTH response to HSA. After colonization with E. coli producing OA, rats tolerant to OA after either 1 or 4 weeks on an OA diet maintained tolerance to OA and bystander suppression HSA. These results suggest that oral tolerance to OA down-regulates signs of local inflammatory response by IgE, IgG antibody and T cell responses to OA, but also provides bystander suppression to an unrelated antigen, HSA.     

The intracellular localization of two antigens after uptake in vivo by peritoneal macrophages from normal mice

Normal mouse macrophages, which had ingested ferritin labelled with fluorescein isothiocyanate and human serum albumin labelled with tetramethylrhodamine isothiocyanate in vivo, were fixed in formalin and embedded for electron microscopy. The examination of sections 1–2 μ thick and adjacent ultrathin sections showed that the yellow-green fluorescent droplets (due to ferritin-FITC) seen by fluorescence microscopy were in the same position as the ferritin-containing phagolysosomes as seen by electron microscopy.

Normal mouse macrophages, which had ingested ¹²⁵I-labelled human serum albumin ([¹²⁵I]HSA) and unlabelled ferritin, were investigated by electron microscopic autoradiography. Both antigens were found to be situated within the same lysosomes.

     

Studies on the relationships between the immunogenicity and catabolism of antigens and their binding to the surface of macrophages

The relationship between immunogenicity of Shigella paradysenteriae, the branched synthetic polypeptide poly-L (Tyr, Glu)-polyL Pro-polyL Lys [(T, G)-Pro–L] and human serum albumin (HSA) interacting with macrophages and the kinetics of antigen degradation and degree of binding to the cell surface was studied. Following thioglycollate inoculation into C57BL/6 mice, the peritoneal-stimulated macrophages had higher levels of hydrolases as compared to unstimulated cells. The lysates of the stimulated macrophages catabolized the three labeled antigens faster than did the lysates of unstimulated cells. However, when degradation of labeled antigens by macrophage cells was assessed, no direct correlation could be demonstrated between the level of cell hydrolases and rate of (T, G)-Pro–L or HSA catabolism. The immunogenicity of the antigens following their uptake by unstimulated and stimulated macrophages was determined by transfer of the antigen-bearing cells into irradiated and nonirradiated syngeneic recipients. No correlation was apparent between the rate of antigen degradation and the capacity to evoke a humoral response. Similarly, no correlation could be demonstrated between the amount of antigen bound to the macrophage cell surface and the immunogenicity of the antigen. It is suggested that neither the rate of antigen catabolism by macrophages nor the amount of antigen bound to the macrophage membrane is the sole factor which determines the immunogenicity of antigens interacting with macrophages.     

The production of specific rabbit antibodies by injecting individual antigen—antibody complexes separated from mixed antigens

Injection of a few hundred micrograms of antigen—antibody precipitates of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme, antigen 3 of Pasteurella pestis and myoglobin into rabbits produced a 10–100-fold increase in antibody compared with that injected in the precipitates. Before injection the precipitates had been separated from either ¹³¹I-labelled human serum albumin serological precipitate or fluorescent ovalbumin serological precipitate by the method of Smith, Tozer, Gallop and Scanes (1962b) after the reaction of mixed antigens with mixed antibodies. The antibodies produced by this method precipitated only their homologous antigen from a mixture of it with either ¹³¹I-labelled human serum albumin or fluorescent ovalbumin. If secondary precipitates formed from antibody produced in this way were injected into rabbits in larger quantities, a further 8–35-fold increase in specific antibody was obtained.     

Fate of Autogenous Canine Lymphocytes after Injection into an Afferent Lymphatic of the Popliteal Lymph Node

Dog thoracic duct lymphocytes were labeled in vitro with ³H-uridine and infused into an afferent lymphatic of the popliteal lymph node of the same dog. Ten minutes after infusion nearly all the injected radioactivity was recovered from the lymph node. An effect of infusion flow rate on the percentage of cells retained by the lymph node was observed ½ to 3½ hours after infusion, and was probably mediated by the tendency of the node to become edematous after infusions at a rate exceeding 0.045 ml/min. Edematous nodes retained 83.7% of the cells, as compared to 47.5% for nonedematous nodes. As early as 30 minutes after infusion a small amount of ³H-radioactivity was found in the spleen and thoracic duct lymph. The deep iliac and paraaortic nodes on the side of the infusion contained significant amounts of ³H-radioactivity, while negligible amounts were detected in the contralateral popliteal node at any time. The intranodal localization of the ³H-labeled cells was studied by radioautography. All labeled cells remained intrasinusoidal during the first 4 hours after infusion. At 9 and 21 hours some labeled cells were located in the extrasinusoidal parenchymal lymphoid tissue of the cortex and the medulla, but the majority still remained intrasinusoidal.     

Legionnaires'' disease serology: Effect of antigen preparation on specificity and sensitivity of the indirect fluorescent antibody test

Sera from 31 Legionnaires' disease (LD) survivors of the Philadelphia outbreak, 31 Legionnaire (L) controls, and 300 additional controls were examined for the presence of specific antibodies to five antigen preparations of Legionella pneumophila (serogroup 1) to determine the effect of antigen preparation on the sensitivity and specificity of the indirect immunofluorescence test. Diagnostic levels were determined for each antigen at the upper limit of normal value (ULNV) titre, which established the titre not exceeded by 85% of controls.

Antigens were prepared from formalin-killed L. pneumophila suspended in egg yolk sac (EYS) (LPF:EYS) or bovine serum albumin (BSA) (LPF:BSA); and from heat-killed organisms suspended in EYS (LPH:EYS) or BSA (LPH:BSA). Antigen was also supplied by the Center for Disease Control (CDC:AG).

Although there was wide variation in the sensitivity of the antigens, at the ULNV level all antigens tested could be used to differentiate LD survivors from L controls (p<0.001; X² test). Formalin treatment resulted in the most specific antigen by eliminating titres in L controls. The results of the X² test, comparing LD survivors with L controls, ranked the antigens in the following ascending order of sensitivity: LPH:BSA 15·3, <CDC:AG 22·8, <LPH:EYS 24·2, <LPF:BSA 45, <LPF:EYS 51. Moreover, when differences in positive results among LD survivors were compared, statistically significant differences were found when LPF:EYS (p<0·01; X² test) and LPF:BSA (p<0·025; X² test) were compared with CDC:AG, LPH:EYS, and LPH:BSA antigens. Titres in 300 additional controls paralleled those found in L controls.

It was concluded that formalin treatment of L. pneumophila resulted in a sensitive antigen, which increased the number of positive tests in survivors while decreasing false-positive tests in controls. It should be considered for use in routine testing programmes for diagnosing LD.