A novel monoclonal antibody recognizing beta(1-3) glucans in intact cells of Candida and Cryptococcus.

The cell walls of all medically important fungi contain a unique polyglucose compound, beta(1-3) glucan. In the present study, murine monoclonal antibodies were produced against linear and beta(1-6) branched beta(1-3) glucans, and their specificities were characterized for reactivity to other beta glucans, fungal cell wall fragments, and fungal cells. Their reactivity was also compared with that of rabbit polyclonal antibodies raised against the same immunogens. Two mouse monoclonal antibodies (AG and BG) recognized immunoreactive epitopes in beta(1-3)(1-6) glucan by ELISA. In an inhibition assay of the anti-beta(1-3)(1-6) activity of the monoclonals, the homologous antigen effectively inhibited the activity as expected, while beta(1-3) also inhibited the assay but to a much lesser extent. No inhibition was obtained by beta(1-3)(1-4) or beta(1-6), while a cell wall extract of Candida albicans (PPM) effectively inhibited both monoclonals. Cell wall fragments of C. albicans (CaCW) and Cryptococcus neoformans (CnCW) inhibited the anti-beta(1-3)(1-6) activity of AG, while BG was much less or not inhibited at all. Immunofluorescence confirmed the unique antibody specificity of AG by its recognition of a beta(1-3)(1-6)-associated epitope on the cell surfaces of C. albicans,C. krusei, C. glabrata, and nonencapsulated C. neoformans. The epitope for the AG antibody is suggested to be present in the branching point of beta(1-3)(1-6), or in the randomly coiled beta(1-3) polyglucan due to the presence of branches. Thus, monoclonal antibodies to beta(1-3)(1-6) glucans may have potential as tools in the laboratory diagnosis of invasive yeast infections.

Circulating beta (1-3) glucan and immunoglobulin G subclass antibodies to Candida albicans cell wall antigens in patients with systemic candidiasis.

Invasive candidiasis in patients who are immunocompromised or in intensive care units (ICUs) presents both diagnostic and therapeutic problems. We previously described antibodies that were directed against Candida albicans cell wall fragments (CW), periodate-treated CW (CW(IO4)), phosphopeptidomannan (PPM), and beta(1-3) glucan. In this study, circulating fungal antigens [mannan and beta(1-3) glucan] and immunoglobulin G (IgG) subclass antibodies to these cell wall antigens (anti-CW) were analyzed in patients with systemic candidiasis. Sera were collected from 14 patients on two or three consecutive occasions, starting on the day when candidiasis was culture proven. The sera were analyzed by enzyme-linked immunosorbent assay. The control groups consisted of lactating mothers (n = 9) (group I) who had breast milk that was positive for C. albicans and also had acute inflammation of the nipples, and age-matched blood donors (n = 10) (group II). Within the first 3 weeks of Candida infection all of the patients were positive for beta(1-3) glucan by the Gluspecy test, but no patients were positive for mannan in the less-sensitive Pastorex Candida test. The controls were negative for both beta(1-3) glucan (<20 pg/ml) and mannan (<2.5 ng/ml). IgG1 anti-CW and IgG2 anti-PPM antibodies were the most discriminatory antibodies. The ratio of IgG1 anti-CW to IgG2 anti-PPM was significantly lower in nonsurviving patients than in the other patients within the first week of candidiasis (P = 0.019). The IgG2 levels of anti-CW(IO4) and antiglucan antibodies correlated strongly (r = 0.681; P < 0.0001), and the absence of these antibodies was associated with increased levels of beta(1-3) glucan. Increased levels of IgG1 anti-CW or IgG2 anti-PPM antibodies (titer of > or = 3 logs) or of a combination of the two antibodies (log sum, > or = 5) showed 92% sensitivity, 100% specificity, and positive predictive values. In conclusion, beta(1-3) glucan and the two subclass antibodies appear to be early specific markers for the laboratory diagnosis of candidiasis. Furthermore, the kinetics of beta(1-3) glucan appearance in serum may assist in evaluating the therapeutic efficacy of antifungal treatments.

Candida albicans cell wall antigens for serological diagnosis of candidemia.

Serological tests for diagnosis of disseminated fungal infections in the immunocompromised host are used with varying results. In the present study, the relative ability of antibodies to specifically recognize Candida albicans cell wall components was evaluated in order to find antigenic markers for serological diagnosis of candidemia. Native C. albicans cell wall fragments (CW), periodate- (CWIO4) and proteinase-K- (CWP) treated CW, a mildly extracted phosphopeptidomannan (PPM), and beta(1-3)(1-6)-glucan were used as antigens in ELISA with sera from rabbits immunized with C. albicans (n = 10), patients with culture proven candidemia (n = 8) and healthy individuals (n = 8). The antibody response in rabbits consisted predominantly of anti-PPM antibodies, a finding that was substantiated by inhibition-ELISA. Consistently, periodate treatment (CW104) destroyed a major proportion of the antigenic epitopes. Low rabbit antibody levels were found against glucan, the major Candida cell wall component. These results supported the conclusion that glucan is localized mainly in the inner part of the C. albicans cell wall. In contrast to rabbits' serum IgG antibody response against PPM, which was at least tenfold higher than that raised against CW, patients with candidemia had similar IgG antibody levels against both antigens. These levels were significantly higher than those seen in healthy controls (CW, P = 0.0005 and PPM, P < 0.0001). Although the human anti-glucan and anti-CWIO4 IgG antibody levels were low overall, they were nonetheless significantly increased in the patient group (P = 0.0159 for antiglucan and P = 0.0491 for anti-CWIO4). In addition, a correlation was noticed between levels of these antibodies. No significant differences were found between patients and controls for IgM antibodies when CW, CWIO4, PPM and Glu were used as antigens. In conclusion, IgG antibodies to PPM and native cell wall fragments (CW) were highly discriminatory for recognition of candidemia and these antigens are thus promising candidates for use in serodiagnosis.

Loss of bactericidal capacity of long-chain quaternary ammonium compounds with protein at lowered temperature.

Amphiphilic betaine esters are quaternary ammonium compounds (QACs) with rapid microbicidal effect, which spontaneously hydrolyze into nontoxic products. thus being referred to as soft antimicrobial agents. The bactericidal effect of 1-decyl (B10), 1-dodecyl (B12), and 1-tetradecyl (B14) betaine esters on Salmonella typhimurium was strongly influenced by temperature, pH and length of hydrocarbon chain. At pH 6.0, presence of 1.5 mM (10% w/v) BSA raised the concentration of B14 for 99% killing (BC2) from 0.006 mM to 1.8 mM. There was a stoichiometric relationship between concentration of BSA and BC2 of B14, indicating that one molecule of B14 was bound per BSA molecule when 99% killing was achieved. When the temperature was lowered to 0 degrees C only minor killing was seen in 1.5 mM BSA at the highest concentration of B14 tested, 57 mM. With B10 at 30 degrees C and pH 6.0, the presence of 1.5 mM BSA raised the bactericidal concentration (BC2) from 0.69 mM to 4.1 mM, and at 0 degrees C and 1.5 mM BSA the BC2 was 11 mM. Thus, the impairment caused of the bactericidal effect of B10 by BSA and lower temperature was less than for B14, since B14 is much more active than B10 at 30 degrees C in the absence of BSA, somewhat more active than B10 at 30 degrees C in the presence of 1.5 mM BSA, and much less active than B10 at 0 degrees C in the presence of BSA. B12 showed properties intermediate between B10 and B14. Lowered pH reduced the bactericidal effect particularly when reduced from pH 5.0 to 4.0 with B10. In the presence of 1.5 mM BSA, the bactericidal effect of 1-dodecyl (DTAB) and 1-hexadecyl (CTAB) trimethylammonium bromide decreased in the same manner as for B10 and B14, respectively. Increasing the time of incubation at 0 degrees C to 50 min, a 99% killing effect was seen with 17 mM CTAB, whereas the same killing effect was reached in 8 min with 17 mM DTAB. Binding of [3H]CTAB to S. typhimurium was also reduced at 0 degrees C in the presence of BSA. Thus, in the presence of 1.5 mM BSA, QACs with the longer hydrocarbon chain were most efficient at 30 degrees C, whereas at 0 degrees C those with the shorter hydrocarbon chain were most active. Consequently, QACs with shorter tails should be used for disinfection in the presence of proteins at lower temperatures.

The effect of hydrocarbon chain length, pH, and temperature on the binding and bactericidal effect of amphiphilic betaine esters on Salmonella typhimurium.

Amphiphilic betaine esters are quaternary ammonium compounds (QAC) with rapid microbicidal action. They are often labeled 'soft antimicrobial agents', since the compounds hydrolyze spontaneously into betaine and fatty alcohols, thus not only losing their surface active properties and toxicity but also becoming amenable to metabolic use. The present results show that the bactericidal effects of 1-decyl (B10), 1-dodecyl (B12), and 1-tetradecyl (B14) betaine esters on Salmonella typhimurium 395 MS decreased with decreasing hydrocarbon chain lengths, decreased at pH below neutral, and were lower at 0 degrees C that at 30 degrees C. At least part of the decreased effect at pH 4.0 as compared to pH 6.0 can be explained by reduced binding. However, reduced binding cannot explain the decrease in the microbicidal effect at 0 degrees C since the binding of B 14 was the same at 0 degrees C and 30 degrees C although 10-30 times higher concentrations were required at 0 degrees C to achieve the same microbicidal effect as at 30 degrees C. Neither can differences in binding explain the great differences seen in microbicidal effect between QAC with different chain lengths. It is proposed that the membrane deformation resulting in killing of S. typhimurium is more efficiently achieved with QAC with longer hydrocarbon chains and that reduced fluidity of the outer membrane of the bacteria at lower temperatures antagonizes the bactericidal effect. Charge interaction seems to be more important for the binding and bactericidal effect for the QAC with shorter hydrocarbon chains. The different effects of pH, temperature, and hydrocarbon chain length on binding, bactericidal effect, and hydrolysis have to be taken into account when optimizing disinfection and the subsequent elimination of disinfectants.

[Advances in the diagnosis of mycoses. New tests detect Candida infections]. / Framsteg inom mykosdiagnostiken. Nya test påvisar Candida-infektioner.

New techniques for the detection of deep fungal infections are reviewed with a focus on Candida albicans, the species most frequently isolated. The introduction provides an outline of the similarities and differences between bacteria and fungi on one hand and animal cells on the other, which are important to bear in mind in the detection and treatment of deep fungal infections. In addition to cultures of biopsy material, blood, and mucous membranes (colonisation), serum tests for such fungal constituents as antigens or beta-glucan, the PCR technique for the detection of fungal DNA, and a test for the D-/L-arabinitol ratio in urine, are all valuable approaches. As yet, no single laboratory test is available that reliably detects disseminated candidosis. Rapid, sensitive, and specific methods for mycological diagnosis need to be developed to minimize the necessity of resorting to empirical therapy when deep mycosis is suspected.

Submicellar complexes may initiate the fungicidal effects of cationic amphiphilic compounds on Candida albicans.

The killing of Candida albicans by a series of amphiphilic quaternary ammonium compounds (QACs) with different hydrocarbon chain lengths was closely related to the binding of the compounds to the cells and damage of the cell membranes. The membrane damage was measured as the level of release of the UV-absorbing material into the medium in which the cells were suspended and as the level of uptake of propidium iodide in individual cells by flow cytometry. It was shown that of the compounds tested, hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide [CTAB]) bound most efficiently. Tetradecyl betainate chloride (B14), tetradecanoylcholine bromide (C14), tetradecyltrimethylammonium bromide (TTAB), and dodecyltrimethylammonium bromide (DTAB) followed and had declining degrees of binding efficiency. The proportion of CTAB bound was almost total at concentrations up to the critical micelle concentration (CMC) of the compound, whereas that of B14 was somewhat smaller. For the two remaining tetradecyl compounds (C14 and TTAB), still smaller proportions were bound at low concentrations, but the proportions rose disproportionally at increasing concentrations to a distinct maximum at concentrations of 0.2 to 0.5 times the CMC. We propose that interfacial micelle-like aggregates are formed at the cell surface as a step in the binding process. An analogous, but less conspicuous, maximum was seen for DTAB. Thus, great differences in the binding affinity of QACs with different hydrocarbon chains at different concentrations to C. albicans were observed. These differences were related to the CMC of the compound. In contrast, the binding of TTAB to Salmonella typhimurium 395 MS was almost total at low as well as high concentrations until saturation was attained, indicating fundamental differences between binding to the yeast and binding to gram-negative bacteria. The importance of lipid-type complexes or aggregates to the antifungal effect of membrane-active substances are discussed.

Susceptibility of lipopolysaccharide-responsive and -hyporesponsive ItyS Mice to infection with rough mutants of Salmonella typhimurium.

The R5 (chemotype Rb) but not the R10 (chemotype Rd) mutant of murine pathogen Salmonella typhimurium 395MS was extremely virulent in intraperitoneal infections of C57BL/10ScCr mice carrying the ityS and lpsD alleles. C57BL/6J (ityS lpsN) and C3H/HeJ (ityR lpsD) mice showed a much higher resistance to the R5 mutant. Further studies were performed with peritoneal macrophages in vitro in order to elucidate susceptibility in lipopolysaccharide (LPS)-hyporesponsive mice carrying ItyS. The intracellular killing capacity of the ItyS LpsD macrophages was lower than that of the ItyS LpsN macrophages for the R5 mutant and may partly explain the increased susceptibility of the ItyS LpsD mice. The deep rough mutant, R10, was rapidly killed intracellularly by the ItyS LpsD macrophages. Processing of the bacteria in macrophages that had phagocytosed R5 or R10 bacteria was followed for up to 18 days by endotoxin measurements (limulus assay) and immunostaining, with monoclonal antibodies to various parts of the LPS molecule being used. Only 0.1% or less of the macrophage-associated bacteria remained alive after 48 h of incubation, and none were alive on day 7. Although immunostaining showed that LPS was present in both the LpsD and LpsN macrophages during the whole incubation period of 18 days, endotoxin activity in the LpsD macrophages on day 7 was lower than that in the LpsN macrophages, indicating that qualitative modifications of the chemical composition or physical state of the LPS molecule occurred. The interleukin-6 response in the ItyS LpsD macrophages was delayed and of shorter duration compared with that in the ItyS LpsN macrophages. The results suggest that the difference between the LPS-hyporesponsive and -responsive ItyS mice in susceptibility to infection with the R5 mutant was due to the lower activation state of the LpsD macrophages during infection, leading to a lower intracellular bactericidal systems of the macrophages. A rapid killing of the bacterium should restrict the infection and may partly compensate for a diminished inflammatory response. The persistence of LPS within the cells is discussed.

Enzymatic hydrolysis of long-chain alkanoylcholines in rat intestinal loops.

BACKGROUND: The hydrolysis of long-chain alkanoylcholines, presumably catalyzed by butyryl-cholinesterase (EC 3.1.1.8), in rat intestinal loops was studied. The substances have earlier been found to be rapidly degraded in vitro. METHODS: Radiolabeled substrates were used, and a radiochromatographic detection method was applied. RESULTS AND CONCLUSION: The long-chain alkanoylcholines were rapidly hydrolyzed. The rates of the reaction and the chain-length dependence were similar to those reported earlier in vitro. At high substrate concentrations the hydrolysis reaction was inhibited. This could be due to conformational changes of the enzyme, caused by the adsorption of the cationic amphiphile, or to a decrease in the free substrate concentration after incorporation of the amphiphilic ester into the lipid layer of the cell membranes. The enzymatic activity towards the substrates in different parts of the rat intestinal tract was also studied and found to be highest in the duodenum.

Long-chain alkanoylcholines, a new category of soft antimicrobial agents that are enzymatically degradable.

A new category of amphiphilic hydrolyzable quaternary ammonium compounds with rapid and high levels of antimicrobial activity was studied. The compounds, alkanoylcholines with hydrocarbon chains of 10 to 14 carbon atoms, are hydrolyzed by butyrylcholine esterase, which is present in human serum and mucosal membranes. The hydrolysis products are common components of human metabolism. Alkanoylcholines were tested and found to be active against gram-negative and gram-positive bacteria as well as yeasts. The microbicidal activities of the alkanoylcholines were comparable to the activities of the stable quaternary ammonium compounds of corresponding chain length and increased with an increasing number of carbon atoms. The compounds were also found to be hydrolyzed by enzymes present in certain microorganisms. The degradation was achieved after reaching the microbicidal effect.

Selective release of the periplasmic enzyme beta-lactamase from Escherichia coli with tetradecyl betainate.

The periplasmic enzyme beta-lactamase was selectively released from Escherichia coli K12 by the amphiphilic quaternary ammonium compound tetradecyl betainate at certain concentration intervals. At low concentrations little enzyme was released, and at high concentrations enzyme inactivation occurred. Greater effects of tetradecyl betainate were seen both with respect to release and inactivation at higher pH. At intermediate concentrations of tetradecyl betainate high yields of beta-lactamase were obtained with no detectable contribution of the cytoplasmic marker beta-galactosidase. The highest yields of beta-lactamase activity were obtained when high concentrations of salt were added 1 min after permeation of the bacteria with tetradecyl betainate.

Butyrylcholinesterase activity towards long-chain alkanoylcholines: kinetics and mechanism.

The hydrolysis of long-chain alkanoylcholines catalyzed by butyrylcholinesterase (EC 3.1.1.8) has been studied. Radiolabelled substrates have been used and a radiochromatographic detection method developed earlier has been applied. The long-chain choline esters were found to be excellent substrates for butyrylcholinesterase at low concentrations, with Km values lower than those of short-chain analogues. At higher substrate concentrations, however, the hydrolysis reaction is inhibited, due to the formation of mixed micelles between the amphiphilic substrate and the corresponding alkanoic acid formed in the hydrolysis reaction. The inhibition may also partially be the result of conformational changes of the protein following adsorption of the cationic amphiphile. Critical micelle concentrations (CMC) for the long-chain substrates, as well as for mixed micelles, have been determined.

Bacterial uptake of octyl ethanolamine increases with pH.

The uptake of octyl ethanolamine (C8EA) by Pseudomonas pseudoalcaligenes was determined at pH 7.1-10.0. At pH 9.1 the total uptake was nearly three times higher and at pH 10.0 four times higher than at pH 7.1. Also the initial rate of uptake was lowest at pH 7.1. At pH 7.1 five to ten times higher concentrations of C8EA were needed than at pH 9.1 to achieve the same degree of leakage of cytoplasmic constituents. The results support the hypothesis that penetration of the bacterial cytoplasmic membrane by C8EA in its uncharged form is favoured. This takes place particularly with high pH in the suspending medium. In the cytoplasm, the pH is lower, and C8EA becomes more protonated. This will prevent back diffusion, promote accumulation and enhance membrane interaction and toxicity at high pH.

Endotoxin shedding by enterobacteria: free and cell-bound endotoxin differ in Limulus activity.

The endotoxin activities of gram-negative bacteria and their lipopolysaccharides (LPS) have been quantitated by a chromogenic Limulus amocbocyte lysate (CLAL) assay. When bacterial cell exposing various cell surface structures were compared, the highest Limulus activities were found in R strains of Escherichia coli and Salmonella typhimurium mutants. E. coli with K antigens did not differ from K-negative strains. By measuring beta-hydroxymyristic acid (3-OH tetradecanoic acid, beta-OHC14:0), it was possible to compare the CLAL activities of LPS bound to bacterial cells, LPS shed into the culture medium, and purified LPS. After 16 h of growth, the cell-free culture supernatants of three E. coli O1K1 strains and S. typhimurium showed CLAL activities 14.3 to 20.3 times higher than did the corresponding bacterial cell suspensions in relation to their beta-OHC14:0 contents. Four other E. coli strains (O serotypes O14, O24, and O75) and the S. typhimurium 395 R mutants MR5 and MR6 showed CLAL values 2.8 to 7.9 times higher in their culture supernatants. LPS of E. coli O1K1 and S. typhimurium had lower CLAL activities than the culture supernatants (1/10 and 1/4, respectively). Although the beta-OHC14:0 concentrations of the culture supernatants were approximately half those of the corresponding bacterial cells, all had CLAL values that were 2 to 21 times higher. The bacterial cell suspension, culture supernatant, and purified LPS of S. typhimurium MS were compared by CLAL assay and a quantitative enzyme-linked immunosorbent assay based on monoclonal antibodies to the O5 antigen. Endotoxin shed into the culture medium was the most CLAL-active form of LPS, while purified LPS was the most antigen-active form. The results emphasize the importance of appropriate standards when quantifying endotoxin in various states. In conclusion, E. coli and S. typhimurium bacteria shed significant amounts of endotoxin into the surrounding medium during growth. This form of LPS is more CLAL active than the cell-bound or purified LPS.

Cure of zygomycosis caused by a lipase-producing Rhizopus rhizopodiformis strain in a renal transplant patient.

A 40-year-old man with renal failure due to membranous glomerulonephritis received a cadaveric renal transplant and immunosuppressive therapy with cyclosporine, azathioprine and steroids. Initially the transplantation was successful. 12 days after the transplantation, however, serous secretion appeared in the wound. Later, black necrosis was seen. Fungal culture showed growth of a zygomycete species. Rhizopus rhizopodiformis, with high in-vitro resistance to amphotericin B, flucytosine, fluconazole, ketoconazole and itraconazole. The MIC value for the allylamine derivative SF86-327 (Exoderil) was 1.6 micrograms/ml. Microscopic examination of sections from a surgical revision showed necrosis of the fat tissue and massive hyphal invasion of the perirenal fat, which contained semi-crystalline material anisotropic as seen in polarized light and characteristically staining with rubeanic acid. These histological data indicate a lipase-induced in-vivo splitting of lipids into fatty acids. In-vitro R. rhizopodiformis showed very high extracellular lipase production. 11 days after initiation of amphotericin B therapy cultures and sections remained positive for rhizopus. Amphotericin B was therefore supplemented with Exoderil orally, cyclosporine and steroids were maintained, and azathioprine was discontinued. The wound granulated, shrank, and healed completely in 10 weeks.

Antimicrobial activity of betaine esters, quaternary ammonium amphiphiles which spontaneously hydrolyze into nontoxic components.

A series of quaternary ammonium compounds that are esters of betaine and fatty alcohols with hydrocarbon chain lengths of 10 to 18 carbon atoms were tested with respect to antimicrobial activities and rates of hydrolysis. When the tetradecyl derivative was tested against some selected microorganisms, the killing effect was comparable to that of the stable quaternary ammonium compound cetyltrimethylammonium bromide. At higher pH values, both the antimicrobial effect and the rate of hydrolysis of the esters increased. However, whereas at pH 6 greater than 99.99% killing of Salmonella typhimurium was achieved with 5 micrograms/ml in 3 min, the rate of hydrolysis was less than 20% in 18 h. At pH 7, a similar killing effect was achieved in 2 min and 50% hydrolysis occurred in ca. 5 h. Thus, it is possible to exploit the rapid microbicidal effect of the compounds before they hydrolyze. The rate of hydrolysis was reduced by the presence of salt. The bactericidal effect of the betaine esters increased with the length of the hydrocarbon chain of the fatty alcohol moiety up to 18 carbon atoms. Since the hydrolysis products are normal human metabolites, the hydrolysis property may extend the use of these quaternary ammonium compounds as disinfectants and antiseptics for food and body surfaces.

Subclass distribution of IgG and IgA antibody response to Pseudomonas pseudoalcaligenes in humans exposed to infected metal-working fluid.

The subclass distribution of the IgG and IgA antibody response in serum was studied in humans exposed to aerosolized metal-working fluid containing Pseudomonas pseudoalcaligenes. This species was consistently found in concentrations of 10(8) bacteria per milliliter of metal-working fluid during 1 year of observation. No increased frequency of respiratory infections or discomfort was related to the exposure to the infected fluid. The antibody response to the bacterium consisted predominantly of IgG1 and IgG2 antibodies. IgG2 antibodies dominated the antibody response to the lipopolysaccharide of the bacterium. IgA1 and IgA2 antibodies were also found. Smokers had significantly reduced antibody levels of all subclasses compared with nonsmokers. The antibody levels in smokers did not differ from levels of the unexposed control group. Analyses of the total serum immunoglobulin concentrations with respect to subclasses revealed that the total IgG2 levels were also significantly reduced in smokers. In nonsmokers, the age of the individuals influenced the antibody levels of the IgG1, IgG2, IgA1, and IgA2 subclasses, the levels decreasing with increasing age. For smokers, the correlation between age and antibody levels was only obvious for IgG2 antibodies. Decreased IgG2 antibody levels in the smokers were also accompanied by decreased FEV1 values (p less than 0.01). Subclass analysis of the antibody response to P. pseudoalcaligenes demonstrated that the subclass pattern for the whole bacterium differed from the pattern of the major cell wall component, the lipopolysaccharide. The significance of qualitative and quantitative differences in the subclass antibody response is discussed.