B7 requirements for primary and secondary protein- and polysaccharide-specific Ig isotype responses to Streptococcus pneumoniae

The requirements for B7 costimulation during an in vivo humoral response to an intact extracellular bacteria have not been reported. In this study we immunized mice with Streptococcus pneumoniae (R36A) to determine the B7 requirements for induction of Ig, specific for two determinants on R36A, the phosphorylcholine (PC) determinant of C-polysaccharide and pneumococcal surface protein A (PspA). We show that the primary anti-PspA response, the development of PspA-specific memory, and the induction of the secondary anti-PspA response in primed mice were completely dependent upon B7 costimulation. Of note, costimulation was required only briefly after the secondary immunization compared with after the primary immunization for optimal induction of Ig. Blockade of B7 costimulation at the time of secondary immunization also completely abrogated the established state of memory, but did not induce tolerance. In contrast to the anti-PspA response, the primary anti-PC response involved only a very short period of B7 costimulation. Whereas B7-2 alone was required for induction of the primary anti-PspA and anti-PC responses, a redundant role for B7-1 and B7-2 was noted for the PspA-specific secondary response. CTLA4Ig blocked both the anti-PC and anti-PspA responses equally well over a wide range of bacterial doses. These studies demonstrate a critical, but variable, role for B7-dependent costimulation during an Ig response to an extracellular bacteria.     

TRF requirements for in vitro PFC responses to SRBC and R36a. I. TRF is distinct from IL 2 but indistinguishable from polyclonal BCSF

In vitro PFC responses to the thymus-independent (TI) antigen Streptococcus pneumoniae R36a require T cell replacing factor(s) (TRF). This requirement for TRF is as significant as for the thymus-dependent (TD) antigen SRBC. TRF is shown to be distinct from IL 2 by the following observations: 1) culture supernatants from the cloned T cell line L2, collected over an 8-day period after allogeneic stimulation, transiently contain IL 2 activity but maintain high levels of TRF activity throughout 192 hr; 2) L2V, a variant subclone of L2, produces much higher levels of TRF activity than the parental line but no detectable IL 2 activity; 3) the addition of IL2+, TRF- supernatants from the T cell hybridoma FS6-14.13 does not affect the L2V SF-driven PFC responses to R36a or SRBC; and 4) the addition of contaminating T cells to cultures containing T cell-depleted spleen cells, L2V SF, and antigen does not affect the PFC response. TRF does appear to be indistinguishable from polyclonal B cell stimulating factor (BCSF), which stimulates polyclonal PFC responses in the absence of antigen, mitogen, or anti-Ig. The TRF and BCSF activities of L2V SF could not be separated by ion-exchange, hydrophobic-interaction, and gel-filtration chromatography. TRF and BCSF have an apparent m.w. of approximately 40,000.     

Regulation of idiotope expression. I. The effect of antigen dose on expression of certain T15 idiotopes during primary IgM response to S. pneumoniae R36a

Clonal heterogeneity among B cells reactive to the same epitope may be determined through differences in idiotypy. It appears that clones bearing distinct idiotopes may constitute functionally distinct subpopulations. Data suggest that idiotopically distinct clones of PC-reactive B cells may be regulated independently of one another. We have looked to see whether individual T15+ clones may also differ in their requirements for activation. Here we examine the effect of immunizing doses of antigen on expression of two T15 idiotopes, B36-82 and B39-38, during both in vivo and in vitro primary responses to Streptococcus pneumoniae R36a (Pn) in CB-20 mouse strain. The idiotopes were detected on the specific antibody plaque-forming cells (PFC) by using monoclonal anti-idiotopic antibodies. We find that distinct patterns of idiotope expression are generated by stimulation with different doses of antigen. Immunization with suboptimal and super-optimal doses of Pn produced responses dominated by PFC expressing both idiotopes, whereas PFC induced by optimal antigen concentrations were primarily B36-82+ and B39-38-. These data indicate that the varying of antigen concentration may induce the response of different B cells bearing distinct idiotypes.     

Both species of chlamydia and two biovars of Chlamydia trachomatis stimulate mouse B lymphocytes

We have investigated the ability of both species of chlamydiae (C. trachomatis and C. psittaci), two major biovars of C. trachomatis (lymphogranuloma venereum and trachoma), and the two developmental forms of chlamydia (reticulate and elementary bodies) to stimulate murine spleen lymphocytes. All of these forms of the bacteria induce potent proliferation and differentiation to plaque-forming cells by B lymphocytes in vitro. Chlamydiae induce a broad antibody response, suggesting that stimulation is polyclonal in nature. Although all chlamydiae possess a lipopolysaccharide (LPS) genus-specific molecule similar to LPS found on Re mutant enterobacteria, polyclonal B cell stimulation is likely caused by molecules other than LPS, since i) polymyxin B failed to inhibit chlamydia-induced immunostimulation and ii) C3H/HeJ mice (LPS nonresponders) produced normal numbers of PFC after culture with chlamydia (but not LPS). Thus, a cross-species moiety that is not LPS is responsible for polyclonal stimulation by chlamydia. Because these bacteria can exist in latent forms in an animal, and all forms are immunostimulatory, the question of whether these bacteria can alter immune responses if released during other infections or immunizations has been raised.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

"Nonspecific" immunoenhancing T cells in tumor-bearing mice include anti-idiotypic subsets

Splenocytes from DBA/2 mice inoculated 3 wk earlier with syngeneic P815 mastocytoma tumor cells produce increased numbers of antibody plaque-forming cells (PFC) when stimulated with either sheep red blood cells (SRBC) or phosphorylcholine (PC) on Streptococcus pneumoniae R36a in vitro. The nature of this nonspecific hyperreactivity was investigated in mixed cultures of purified splenic T and B cells. The addition of T cells from P815 tumor-bearing mice (TP815) into the cultures of normal B cells produced a significant enhancement of the PFC response to both SRBC and PC, when compared with the effect of normal T cells added to control cultures. The idiotypic profile of the enhanced anti-PC response was studied by a PFC-inhibition assay with monoclonal antibodies against two distinct idiotopic determinants (Id) of the T15 family. Normal B cells produced greater than 90% of T15 Id-positive (Id+) PFC. Addition of normal T cells diminished the proportion of T15 Id+ PFC to approximately 60%, whereas the rest of PFC were Id-. Addition of the immunoenhancing TP815 cells into the normal B cells cultures elevated the number of both T15 Id+ and Id- PFC responses, proportionally. However, when TP815 cells were first incubated on T15 protein-coated dishes and the non-adherent fraction was added to B cell cultures, the anti-PC PFC response remained enhanced but consisted of predominently T15 Id- PFC. These observations suggest that the early stage of P815 tumor growth activates various populations of specific helper/amplifier T cells including subsets with anti-idiotypic activity and that the generalized increase of antibody response to various antigens in tumor-bearing mice may be regarded as a polyclonal activation of specific T cells.     

An examination of the TRF assay reveals a heterogeneity of TRF-like activities

Supernatant from the cloned Mlsa,d-reactive helper T cell line L2V is a potent source of T cell replacing factor (TRF) activity. To determine whether L2V SF was representative of TRF-active supernatants, it was compared with supernatant from Con A-stimulated spleen cells (CASF) by using TRF assays. This analysis, facilitated by the use of four different antigens (R36a, TNP-R36a, SRBC, and HRBC), produced the following results. 1) L2V SF and CASF allowed responses of similar magnitude against either R36a or TNP-R36a; however, CASF always allowed responses of fivefold to 30-fold greater magnitude against SRBC than L2V SF. 2) B cells from xid and "normal" mice will give equally large responses to TNP-R36a when CASF is used as the source of TRF; however, L2V SF will only effect the responses by "normal" B cells. 3) L2V SF-driven responses to R36a and SRBC, and CASF-driven responses to R36a, follow single-hit kinetics and are IL 2 independent, whereas CASF-driven responses to SRBC follow multi-hit kinetics and are IL 2 dependent. These results indicate that the TRF assay measures a heterogeneity of TRF-like activities that can be distinguished according to the supernatant, antigen, and/or B cell responders used. The determination of whether this heterogeneity is due to the number of molecular components of a single "type" of TRF required for each antigen-induced PFC response or to the existence of distinct "types" of TRF is examined.     

The repertoire diversity and magnitude of antibody responses to bacterial antigens in aged mice: I. Age-associated changes in antibody responses differ according to the mouse strain

Aging influences the host immune responses in various ways. In aging mice we have studied the antibody responses to two unrelated bacterial antigens. Streptococcus pneumoniae R36a vaccine (Pn) and TNP coupled to Brucella abortus (TNP-BA). Aged animals (20-24 months old) of the C57BL/6 strain had markedly reduced numbers of IgM antibody plaque-forming cells (PFC) to Pn as compared to young/adult mice (2-3 months old). In contrast, the anti-Pn IgM PFC responses of aged BALB/c mice were consistently higher than they were in the young/adult mice. The increased anti-Pn responses were not due to a nonspecific immunostimulation, because the responses of aged BALB/c mice to TNP-BA were lower as compared to the adults. However, the aged BALB/c mice responded relatively poorly to Pn challenge, and their IgG responses (as determined by ELISA plaque assay) demonstrated a very high individual variability. The clonotypic diversity of anti-Pn response in young BALB/c and C57BL/6 is limited, such that the majority of PFC produce antibody that express all idiotopes (Id) of the T15 immunoglobulin encoded in the VH-S107/Vk22 genes. In contrast, the PFC from aged mice are diverse, expressing incomplete T15 Id or none at all, suggesting that the antibodies are encoded by altered T15 genes and by different, non-T15 genes. Our data demonstrate that the age-related changes in the magnitude of antibody response to certain antigens are influenced by the host genetic make-up, and that the changes in magnitude and diversity of antibody response may be unrelated to each other.     

Inhibition of antibody responses to phosphocholine by C-reactive protein

C-reactive protein (CRP) is an acute phase serum protein in man that binds to the cell wall C-polysaccharide (PnC) of Streptococcus pneumoniae via phosphocholine (PC) determinants. We have previously shown that in mice CRP increases splenic clearance of PnC-coated autologous erythrocytes and S. pneumoniae, and increases survival after pneumococcal infection. Because CRP alters clearance of particulate PnC antigens, we tested its effect on immunization with pneumococci. Pretreatment of mice with 50 to 200 micrograms CRP 30 min before immunization with serotype 3 S. pneumoniae resulted in dose-dependent inhibition of the antibody response to PC. Both serum hemagglutinin and splenic PFC against PC were decreased in CRP-treated mice tested from 1 to 10 days after injection of antigen. CRP treatment had no effect on the antibody response to the serotype 3 capsular polysaccharide, another T-independent antigen. To determine whether CRP inhibition was related to altered processing of particulate antigen, mice were immunized with horse red blood cells (HRBC) conjugated with PC or PnC and the PFC responses to PC and HRBC were determined. CRP treatment resulted in specific inhibition of the PFC response to PC in both cases without affecting the response to HRBC. These results indicate that inhibition of the antibody response by CRP is not the result of altered antigen localization and processing, and that CRP may prevent immunization by masking determinants on bacterial or other surfaces.     

Pregnancy-associated growth factor. II. A T-dependent polyclonal activator of human adult peripheral blood lymphocytes (PBL)

This study examined the ability of pregnancy-associated growth factor (PAGF), a substance found in crude human chorionic gonadotropin (hCG), to induce plaque-forming cells (PFC) in cultured human peripheral blood lymphocytes (PBL). PAGF, 0.25 to 1 mg/ml, induced maximal PFC at 6 to 7 days as measured by the staphylococcal protein A-coupled SRBC reverse hemolytic plaque assay with a rabbit anti-human Ig antiserum. PAGF-induced PFC/culture ranged from 1800 to 39,000 with a mean of 11,524 in unfractionated PBL (N = 24), as compared to 540 to 77,840 with a mean of 17,303 for pokeweed (PWM) (N = 22). Comparison of PAGF- and PWM-induced PFC showed that both induced specific IgG, IgA, and IgM PFC. In most individuals, PAGF induced more IgM and PWM more IgG PFC. The kappa: lambda ratio was 1.5 for unstimulated PBL, and approximately 3.5 for PAGF and PWM. To see if PAGF was a T-dependent polyclonal activator of B cells, T and non-T populations were obtained by SRBC rosettes and negatively selected T4 and T8 cells by complement-mediated lysis of SRBC+(T) cells. Only the recombined subsets which included T4 cells and non-T cells supported PAGF- and PWM-induced PFC. These data indicate that PAGF, a substance derived from commercial extracts of pregnancy urine, is a T4-dependent polyclonal activator of normal human B cells.     

C-reactive protein enhances immunity to Streptococcus pneumoniae by targeting uptake to Fc gamma R on dendritic cells

C-reactive protein (CRP) is an acute phase reactant with roles in innate host defense, clearance of damaged cells, and regulation of the inflammatory response. These activities of CRP depend on ligand recognition, complement activation, and binding to FcgammaR. CRP binds to phosphocholine in the Streptococcus pneumoniae cell wall and provides innate defense against pneumococcal infection. These studies examine the effect of this early innate defense molecule on the development of Abs and protective immunity to S. pneumoniae. Dendritic cells (DC) initiate and direct the adaptive immune response by integrating innate stimuli with cytokine synthesis and Ag presentation. We hypothesized that CRP would direct uptake of S. pneumoniae to FcgammaR on DC and enhance Ag presentation. CRP opsonization of the R36a strain of S. pneumoniae increased the uptake of bacteria by DC. DC pulsed with untreated or CRP-opsonized R36a were transferred into recipient mice, and Ab responses were measured. In mice challenged with free R36a, CRP opsonization resulted in higher secondary and memory IgG responses to both phosphocholine and pneumococcal surface protein A. Furthermore, mice immunized with DC that had been pulsed with CRP-opsonized R36a showed increased resistance to intranasal infection with virulent S. pneumoniae. The effects of CRP on Ag uptake, Ab responses, and protection from infection all required FcR gamma-chain expression on DC. The results indicate that innate recognition by CRP enhances effective uptake and presentation of bacterial Ags through FcgammaR on DC and stimulates protective adaptive immunity.     

Streptococcal serotype carbohydrate represents a novel class of type 2 antigen which is T-independent

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyclonal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb-3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.     

Induction of antigen-specific antibody responses in primed and unprimed B cells. Functional heterogeneity among Th1 and Th2 T cell clones

CD4+ T cells have been recently divided into two subsets. The functions of these subsets are thought to be distinct: one subset (Th1) is responsible for delayed type hypersensitivity responses and another (Th2) is primarily responsible for induction of antibody synthesis. To more precisely define the roles of both subsets in humoral immune responses, we examined the ability of a panel of nominal antigen specific Th1 and Th2 clones to induce anti-TNP specific antibody synthesis in TNP-primed or unprimed B cells. Four of nine Th1 clones induced little or no antibody synthesis with TNP-primed B cells. However, five other Th1 clones were very effective at inducing IgG anti-TNP plaque-forming cell (PFC) responses in primed B cells. One of these Th1 clones was analysed in detail and found to also provide helper function for unprimed B cells. Cognate B-T cell interaction was required for induction of both primary and secondary responses with this clone, indicating that a Th1 clone could function as a "classical" Th cell. The seven IL-4 producing Th2 clones examined were also heterogeneous in their ability to induce antibody secretion by TNP-primed B cells. Although four of the Th2 clones induced IgG and IgM anti-TNP PFC responses, two Th2 clones induced only IgM and no IgG antibody, and another clone failed to induce any anti-TNP PFC. All Th2 clones failed to induce any anti-TNP PFC. All Th2 clones produced high levels of IL-4, but "helper" Th2 clones produced significantly greater amounts of IL-5 than "non-helper" Th2 clones. These studies indicate that some IL-2- and some IL-4-producing T cell clones can induce TNP-specific antibody in cell clones can induce TNP-specific antibody in primed and unprimed B cells, and that Th1 and Th2 clones are heterogeneous in their ability to induce Ig synthesis. Therefore, although T cell clones can be classified as Th1 or Th2 types according to patterns of IL-2, IFN-gamma, or IL-4 synthesis, the functional capacity to induce antibody synthesis cannot be predicted solely by their ability to secrete these lymphokines.     

Polyclonal B Cell Activation by Cell Wall Preparations of Gram-Positive Bacteria

The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram-positive bacteria were studied using the anti-sheep red blood cell (SRBC) or anti-trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS-responder), C3H/HeJ (LPS-non-responder), (CBA/N × Balb/c) F1 male with an X-linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA-ability of synthetic peptidoglycan, muramyl dipeptide (N-acetylmuramyl-L -alanyl-D -isoglutamine, MDP), was also examined, and a similar activity was observed in MDP.     

B-cell subsets responsive to fluorescein-conjugated antigens: II. “Cross-priming” and its elimination by bromodeoxyuridine and light

In vitro stimulation of normal splenic B cells with a variety of fluorescein (FL)-conjugated immunogens stimulated the incorporation of bromodeoxyuridine to the extent that all subsequent FL-immunogen-induced PFC responses were inhibited after exposure to light. The effect was hapten specific and suggests that any FL-immunogen can trigger an initial wave of DNA synthesis independent of surface IgD or polyclonal B-cell activating receptor interaction.     

The immunoregulatory role of bone marrow. IV. Role of an immunoenhancing glycoprotein derived from murine bone marrow

Bone marrow-enhancing factor (B-EF) is the spontaneous product of whole bone marrow cells cultured in serum-free medium for a short term (24-48 hr). The factor is prepared by ultrafiltration of BMC supernates to yield a preparation with a MW of greater than 10,000. Production of the factor is not dependent upon antigenic or mitogenic stimulation of BMC, but is inhibited by treatment of BMC with cycloheximide. B-EF augments the in vitro primary PFC response to SRBC, as well as in vitro secondary IgM and IgG PFC responses to SRBC. Enhancement by B-EF is antigen dependent, genetically nonrestricted, and maximal when present at the initiation of culture. B-EF cannot induce a polyclonal antibody response like the polyclonal activator LPS. B-EF is directly mitogenic for thymocytes, bone marrow, and whole spleen cells, but fails to act as a costimulator of thymocyte proliferation in the presence of Con A. B-EF cannot support the growth of the IL-2-dependent cell line CTLL-2. Since B-EF has not been purified, the supernatant may contain more than one activity. The factor is heat labile at 65 degrees C and is sensitive to enzymatic digestion with trypsin and neuraminidase; this implies that B-EF may be a glycoprotein.     

Studies of immune responses in mice prone to autoimmune disorders. II. Decreased down-regulation by auto-anti-idiotype antibody in autoimmune-prone mice

Three lines of evidence are presented which suggest that autoimmune-prone mice are deficient in the production of auto-anti-idiotype antibody during their immune response to trinitrophenylated Ficoll (TNP-F). NZB, MRL lpr/lpr and older BXSB male mice have no hapten-augmentable plaque-forming cells (PFC). Hapten-augmentable PFC have been previously shown to be cells whose secretion of antibody has been inhibited by the binding of auto-anti-idiotype antibody to cell surface idiotype. Sera from TNP-F immunized NZB mice lack PFC inhibiting activity (anti-idiotype antibody). Spleen cells from TNP-F immune NZB mice fail to transfer anti-idiotype antibody-mediated suppression to naive mice as do spleen cells from immune non-autoimmune-prone mice. Taken together these data suggest that autoimmune-prone mice are deficient in auto-anti-idiotype antibody-mediated downward regulation of their immune responses. It was further shown that the immune response of NZB mice to TNP-F shows a slower decline in splenic PFC and a greater heterogeneity of PFC affinity than do the responses of non-autoimmune-prone strains. Since athymic (nude) mice, which were previously shown to be defective in the production of auto-anti-idiotype antibody, also show a slower decline in splenic PFC and an increased heterogeneity of PFC affinity, it is suggested that these peculiarities of the immune responses of autoimmune-prone and athymic mice are also the consequences of the lack of auto-anti-idiotype antibody-mediated down-regulation.     

Induction of monocytic suppression after stimulation of peripheral human mononuclear cells with staphylococcal protein A and Staphylococcus aureus

Staphylococcal protein-A (SPA) and Staphylococcus aureus are known to be polyclonal human B-cell activators. It was noted that they induced plaque-forming-cell (PFC) responses lower than those induced by pokeweed mitogen (PWM) and the possibility of early triggering of a suppressor cell was investigated in the present series of experiments. Peripheral mononuclear cells (MNC) were passed through Sephadex G-10 columns to eliminate monocytes. The PFC responses to SPA and S. aureus were thereby increased. PWM-driven PFC responses are suppressed by the simultaneous presence of SPA in a dose-related way, if present in the early phases of the cultures. MNC precultured with SPA or S. aureus have the ability to suppress the PFC response of autologous MNC to PWM. Interestingly this suppressor cell activity was radiation resistant and could not be abrogated by treatment with anti-T-cell monoclonal antibody plus complement. The above experiments clearly demonstrate that the observed low PFC responses of MNC after stimulation with SPA and S. aureus are due to the induction of suppressor cells by these stimulants. The suppressor cells are apparently of monocytic origin.     

A perfluorochemical loss/restoration (L/R) system for tidal liquid ventilation

Tidal liquid ventilation is the transport of dissolved respiratory gases via volume exchange of perfluorochemical (PFC) liquid to and from the PFC-filled lung. All gas-liquid surface tension is eliminated, increasing compliance and providing lung protection due to lower inflation pressures. Tidal liquid ventilation is achieved by cycling fluid from a reservoir to and from the lung by a ventilator. Current approaches are microprocessor-based with feedback control. During inspiration, warmed oxygenated PFC liquid is pumped from a fluid reservoir/gas exchanger into the lung. PFC fluid is conserved by condensing (60-80% efficiency) vapor in the expired gas. A feedback-control system was developed to automatically replace PFC lost due to condenser inefficiency. This loss/restoration (L/R) system consists of a PFC-vapor thermal detector (+/- 2.5%), pneumatics, amplifiers, a gas flow detector (+/- 1%), a PFC pump (+/- 5%), and a controller. Gravimetric studies of perflubron loss from a flask due to evaporation were compared with experimental L/R results and found to be within +/- 1.4%. In addition, when L/R studies were conducted with a previously reported liquid ventilation system over a four-hour period, the L/R system maintained system perflubron volume to within +/- 1% of prime volume and 11.5% of replacement volume, and the difference between experimental PFC loss and that of the L/R system was 1.8 mL/hr. These studies suggest that the PFC L/R system may have significant economic (appropriate dosing for PFC loss) as well as physiologic (maintenance of PFC inventory in the lungs and liquid ventilator) impact on liquid ventilation procedures.     

Specific suppression of the antibody response in vitro by serum from paralyzed mice.

BALB/c mice immunized with either the whole vaccine or the C-polysaccharide obtained from the R36A strain of pneumococcus produce antibody to phosphorylcholine. Mice injected i.v. with a single high dose of the C-polysaccharide are specifically unresponsive to immunization to phosphorylcholine for many months and are considered paralyzed. The induction of paralysis does not eliminate cells reactive to phosphorylcholine; however, serum from paralyzed mice specifically suppresses the response of cultures of normal spleen cells to phosphorylcholine. Paralyzed mice have an early low antibody response to phosphorylcholine and to the receptor for phosphorylcholine as indicated by plaque-forming cell assays. The factor or factors present in serum which may suppress cultures, and, by presumption, be responsible for paralysis are complexes of antigen, antibody, and antibody to the receptor for phosphorylcholine.