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Pieces of human skin from the skin bank were heated in an autoclave for 1 or 5 min at temperatures 80, 90, 100, 110 and 135 degrees C. The pieces were then homogenized and the homogenates were injected intraperitoneally into groups of mice. The amount injected was either a quantity equivalent to 50 or 75% of the mouse body surface area. Fourteen separate experiments were carried out, each one with a variety of temperatures. Mortality in the groups of mice was recorded by the 8th day. Control mice received homogenates of skin heated to no more that 38 degrees C and out of a total of 104 control mice there were only 4 deaths. In contrast homogenates of skin heated to 135 degrees C killed from 80 to 100% of the mice in different groups, averaging 92%. Skin heated to 110 degrees C killed from 33 to 90% of the mice in different groups, averaging 63%. Skin heated to 100 degrees C killed from 0 to 80% of the mice in different groups, averaging 33%. Temperatures of 80 and 90 degrees C killed no more than 10% of the mice in any group, averaging less than 3%. One minute of heating seemed to be sufficient to induce the toxic effect in the skin. These findings indicated that wet heat application to skin was capable of inducing toxicity in a fashion similar to that demonstrated many years ago with hotter dry temperatures applied to skin for 15s. That application was shown to induce polymerization of skin cell membrane lipid proteins rendering them toxic. In this study, increasing toxicity appeared similarly to depend on the quantity of wet heat input as illustrated by the range of increasing temperatures. The relatively lower temperatures of scalding versus flame burns can accomplish similar dangerous effects; it is simply a quantitative matter of heat input.

The burden on military medical services in handling burn casualties is daunting as all physiological systems become affected. Severe burns in a battlefield setting have a very low salvage rate, to a great degree because of the immune failure which invariably develops. Evaluations of responses of lymphocytes taken from burn patients over several weeks following the burn (> 30% total burn surface area), have revealed that the immune failure which follows thermal injury involves T-cell activation events. Interleukin 2, which is normally produced by activated T lymphocytes, is very poorly produced by cells cultivated in vitro taken from non-surviving patients, whereas some production continues, although at below normal levels, in patients who ultimately survive their injury. IL2 exogenously added to lymphocyte cultures enhances the proliferation of cells from surviving patients but gives no such help to cells from non-survivors. The TAC portion of the IL2 receptor (IL2R alpha), expressed on the T-cell surface, appears to be responsible for this difference, as the number of lymphocytes able to express IL2R alpha falls post-burn. A lipid protein complex (LPC) produced in skin by burning has been shown to inhibit the immune response in vivo and the growth of IL2-dependent lymphocytes in culture. Cerium nitrate, applied topically to the burn patient, is thought to fix the LPC in the burn eschar and prevent its entry into the circulation. In a study of ten patients, bathed in cerium nitrate, some T-lymphocyte activities were found to be in the normal range rather than suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)

In the immunosuppressed burn patient serum levels of both IL-2 and a soluble form of IL-2 receptor alpha (sIL-2R alpha) are significantly elevated. Strikingly, the production of these markers by the in vitro activated patients' cells is decreased. This study examines the role of IL-2 in the decreased production of the sIL-2R alpha in vitro in patients with major burns (n = 18, 30 to greater than 70% total body surface area). Peripheral blood mononuclear cell (PBMC) cultures from patients with highly elevated serum sIL-2R alpha, and from healthy controls (n = 12) were activated with concanavalin A (Con A) at initiation. In patients' cultures mitogen-induced increments of sIL-2R alpha levels were significantly lower. There was a significant negative correlation (r = 0.64, P less than 0.001) between a high serum sIL-2R alpha level and a decreased lectin-induced sIL-2R alpha release in vitro. Low levels of sIL-2R alpha in patients' samples were not normalized by increasing the number of T lymphocytes. Also exogenous rIL-1 was without effect, whereas rIL-3 increased sIL-2R alpha release in some cultures. However, sIL-2R alpha levels were significantly increased in patients' cultures by (i) addition of exogenous IL-2; (ii) removal of adherent cells; (iii) addition of cyclooxygenase inhibitor, indomethacin; (iv) bypassing cell surface activation by the combination of the calcium ionophore A23187 and the phorbol ester 12-o-tetradecanoyl acetate. The cyclic AMP-elevating drug, forskolin, abrogated the ability of exogenous IL-2 to increase sIL-2R alpha production. Thus, in the burn patient, the reduced in vitro sIL-2R alpha release appears to relate to abnormalities in IL-2 production and action mediated through its functional surface receptor. Elevated levels of sIL-2R alpha in vivo may, therefore, reflect systemic activation of T lymphocytes in response to biologically active IL-2.

The capacity to induce immunoglobulin (Ig) secretion by soluble T-cell-replacing (TCR) factors derived from alloantigen-stimulated T lymphocytes of blunt trauma patients (n = 15, mean ISS = 24) was examined in Staphylococcus aureus (SAC)-activated normal B-cell cultures. The majority of the patients studied demonstrated a profound suppression of the T-cell-dependent, pokeweed-mitogen-induced Ig production. However, the activity to induce Ig secretion associated with TCRs from the same patients was not reduced compared with that of TCRs from normal subjects. IgM synthesis was normal and IgG secretion induced by TCRs was within the control range (in 6 of 15 patients) or significantly higher (p less than 0.05) than that in the remaining patients. Both patient-derived and control TCRs failed to induce Ig synthesis in cultures of resting B cells and had comparable mitogenic effects on normal SAC-activated and phytohemagglutinin A-activated B and T lymphocytes, respectively. Thus, the intrinsic ability of T lymphocytes to produce B-cell helper factors appears to be unaffected following blunt trauma. Suppression of the T-cell-regulated Ig secretion in traumatized patients may therefore reflect an altered B lymphocyte response to such factors.

Sixty-four patients aged 16-74 years with total body surface area burns (TBSA) ranging from 30 to 90 per cent, were given one bathing in 0.04 M cerium nitrate within 4 h of admission to hospital. Of 21 patients aged 16-30 years, one died (aged 28 with 90 per cent TBSA), and of those aged 31-74 years, two died, one (aged 50 years with 55 per cent TBSA) had multiple internal injuries, the other (aged 51 years with 55 per cent TBSA) had a pulmonary embolism at day 19. Two risk scores, developed from data on 11,200 burn patients treated by standard methods (Roi et al. 1983), were applied to the analysis of risk for 59 patients for whom both total burn surface (TB) and full thickness (FT) areas had been recorded. About 20 patients bore risk of 0.8 or greater on the FT scale and 1.0 on the TB scale, yet instead of 80 per cent deaths among these, only two died. No FT assessment had been made on the multiple injury death whose TB risk score was 0.66. Such survival results in high-risk patients should encourage the use of cerium nitrate for treating serious burn injury.

A general consensus that thermal injury affects T lymphocyte function adversely is supported particularly by the observation that burned patients' lymphocytes secrete reduced levels of biologically active IL-2 in vitro. In the same patients, however, high serum concentrations of the low-affinity IL-2 receptor (IL2R alpha), a product of an IL-2-activated gene, have been observed. In this study a significant proportion of patients also demonstrated over-physiological levels (from 2 to 500 U/ml) of serum IL-2 ascertained by immunoassay. Increases in serum IL-2 content correlated significantly (P less than 0.02) with those of serum IL-2R alpha during the first week post-burn. Later, serum IL-2R alpha levels continued to increase up to 30 days while IL-2 eventually declined. Thus, augmented secretion of IL-2R alpha appears related to the high serum IL-2 content. Therefore refractoriness to further immune stimulation may be due to early activation of the lymphoid system, rather than to an intrinsic incapacity of T lymphocytes for generating sequential responses.

The lipid-protein complex (LPC) formed by thermal injury to skin, which has been shown to have a toxic effect on mice, and which suppresses the immune response, was tested for its specific influence on monocytes. Growth of bacterial endotoxin-stimulated peripheral blood mononuclear cells (PBMC) was inhibited in the presence of LPC, however, the inhibition was less at the time of the optimal rate of cell proliferation. Inhibition was proportional to LPC concentration. ConA-stimulated PBMC were also inhibited by LPC in a dose-related manner. PBMC, in the presence of LPC, secreted interleukin 1 (IL1) at an increasing rate as LPC concentration rose from 5 to 40 micrograms/ml, and the levels of IL1 which could be induced by endotoxin were increasingly amplified in the presence of LPC. In comparison to LPC, the native tissue proteins which were isolated from unburned skin by the same techniques which produced LPC from burned skin, were tested for their effect on PBMC. Native proteins had no effect on IL1 secretion, whether on background or endotoxin-stimulated levels. Thus, the thermally induced change in skin proteins has a specific effect on monocyte IL1 secretion which is not matched by the native proteins, indicating that burn injury to skin specifically affects the lymphokine cascade and consequent immune function.

This study examines the relationship between the capacity for interleukin-2 (IL2) production and the magnitude of the in vitro and in vivo secretion of IL2R alpha in 43 patients with major burns (30-90 per cent total body surface area). Throughout the postburn period a significant (P less than 0.001-0.05) proportion of patients studied demonstrated increasingly high levels of serum IL2 ranging from 2 to over 500 U/mL. Serum IL2R alpha also increased, reaching its highest levels at 15-40 days postburn, while serum IL2 gradually declined. In this period in vitro IL2 production and IL2R alpha secretion in patient's cultures were significantly reduced compared to control. However, in parallel cultures supplemented with exogenous IL2, IL2R alpha levels could be significantly increased (2.5 fold). IL2R alpha levels also approached normal in peripheral blood mononuclear cell cultures from recovering patients whose in vitro IL2 production had improved. These observations suggest that in the burn patient altered synthesis and/or secretion of the soluble form of IL2R alpha may be related to IL2 content. Above physiological levels of IL2R alpha and its ligand in postburn serum also indicate that thermal injury induces strong in vivo activation of the lymphoid system.

Normal peripheral blood mononuclear cells (PBMC) were incubated with the lectins PHA and ConA to stimulate IL2 release into the culture supernatants. In the added presence of the lipid-protein complex (LPC) derived from burned skin, PHA and ConA produced much less bioavailable IL2, the combination with PHA being more inhibitory of its production than that with ConA at concentrations of 1 microgram and 5 micrograms lectin/ml. As LPC alone also elicited IL2 production the inhibition of active IL2 production with these lectins was seen as a synergistic reaction with LPC. This was not altered by incubating cells with PHA alone, followed later by LPC, suggesting that LPC affects later molecular events which develop in T-cell activation. However, after incubating LPC first and washing it from the cells, both lectins were able to stimulate secretion of higher levels of bioavailable IL2, but again, less IL2 was produced with PHA than with ConA. Since PHA and ConA are reported to react with the T-cell receptor (TCR) and CD3 T-cell surface antigens, respectively, although both react additionally with CD2, it appears that LPC interfered more directly with TCR-related reactions than those involving CD3, although the two antigens have been considered to be interdependent. LPC is a trimer of a complex of six proteins from skin cell membranes, which had coalesced under the influence of thermal energy. The six proteins have relative molecular weights of 40, 50, 65, 110, 120 and 160 kDa. By coincidence 40 kDa and 51 kDa are the weights of the heterodimer subunits of TCR alpha/beta, and CD2 is 50 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid peroxides, formed as a consequence of oxygen free radical formation, are responsible for tissue damage in a great variety of pathological conditions including thermal injury. 'Cutaneous burn toxin', formed by application of heat to skin, is thought to be specific to the burn injury. It causes dose-dependent damage to mitochondrial and red cell membranes, and dose-dependent inhibition of interleukin-2-dependent growth of lymphocytes. The possibility that the toxicity of 'cutaneous burn toxin', a lipid-protein, is exerted through lipid peroxides, was examined by measuring the levels of both agents in plasmas of eight burn patients during the first week after their injury. It was observed that plasma lipid peroxides did not appear in parallel with absorption into the circulation of 'cutaneous burn toxin'. Lipid peroxide levels equally common to very low and very high burn toxin levels, were recorded. The pair of agents correlated negatively (r = -0.26) at a significance of only 0.1. In addition, isolated purified 'cutaneous burn toxin' contained no measurable lipid peroxide. No relationship was therefore demonstrated between plasma levels of 'cutaneous burn toxin' and lipid peroxides.

Endotoxins of E. coli, S. typhosa and Ps. aeruginosa were injected i.p. into mice a few days before administration of the antigen sheep erythrocytes (SE). Antibody-forming cells (AFC) to SE were later enumerated in relation to dose of endotoxin given. In comparison a toxic lipid protein isolated from burned skin (cutaneous burn toxin or CBT) was similarly applied and found to be more inhibitory of the immune response than any of the three endotoxins. Considering the 50 per cent inhibitory doses on a molar basis CBT was found to be 1000 fold more immunosuppressive than the most inhibitory endotoxin. As the immune suppression which follows severe thermal injury involves failure of interleukin 2 (IL2) function, as a critical index of survival, the CBT was tested for its effects on the culture of a human IL2-dependent cell line in the presence of IL2. CBT inhibited the growth of these cells, however, endotoxin had no effect on their proliferation. Thus CBT, which arises by a thermally induced polymerization of skin lipid protein, is specific to burn injury and has a direct inhibitory effect on the immune response.

Spontaneous and pokeweed mitogen (PWM)-induced polyclonal immunoglobulin production in vitro were estimated in patients with severe burns (28-83% TBSA) immediately following their injury and at intervals of 8 to 14 days thereafter. In parallel, functional activity of patients' T lymphocytes was assessed in the mixed lymphocyte reaction (MLR). PWM-induced immunoglobulin secretion, of both IgG and IgM, was elevated in the first 2 to 3 weeks after burn, followed by a period of relatively suppressed antibody response at 3 to 4 weeks. Immunoglobulin production was then apparently restored to baseline or higher levels in surviving patients. However, in patients whose antibody response continued to be suppressed fatal septicemia developed. The background (spontaneous) immunoglobulin synthesis increased significantly over the normal range in 10/78 tests performed. These alterations in mitogen-induced immunoglobulin production were well correlated with changes in the T-cell alloreactivity in the MLR. Thus thermal injury-associated changes in the patients' humoral responses may be a consequence of changes within the T-cell compartment, especially the T-helper cell subset.

In vivo resistance of mice to Neisseria meningitidis was entirely abrogated by a concomitant administration of mucin and iron with N. meningitidis organisms. Resistance, however, was restored when the latter challenge was given to animals which had been immunized 7 days previously with a crude extract of meningococcal antigens (MA), BCG, or proteose peptone. These results suggest that depression or activation of the reticuloendothelial system (RES) may be important in resistant of mice to meningococcal infection. Also, like BCG, MA inoculation was able to prevent infection by Listeria monocytogenes indicating its marked ability to activate the RES. The data show that immunization can induce nonspecific RES stimulation and that the nonspecific resistance persists for at least 7 days.

IL2 secretion in response to the T-cell mitogen Staphylococcal protein A (SPA) is significantly decreased in patients with major burns (n = 10, greater than 20% total body surface area) up to 50 days postburn in comparison with normal control (greater than 2 to 10 U/ml and 16 to 36 U/ml, respectively). Activation of protein kinase C (PK-C) and changes in [Ca++]i are both normally implicated in the production of IL2. Bypassing the requirements for mitogen-induced increases in [Ca++]i, using the cation ionophore A23187, or activating PK-C with the phorbol ester 12-o-tetradecanoyl-phorbol-13-acetate (TPA), failed to significantly restore SPA-induced IL2 production in cell cultures from burned patients. The combination of A23187 and TPA significantly (p less than 0.005-0.001) enhanced IL2 secretion in patients' cell cultures (range, 20 to greater than 64 U/ml). However, the levels of IL2 from the burned patients' cultures remained significantly lower (p less than 0.05) than those in control cultures exposed to TPA and A23187 (range, 256 to greater than 600 U/ml). Therefore, the burn-related defect in mitogen-induced IL2 secretion is only partially bypassed with cation ionophores and phorbol esters. This suggests that the abnormality in IL2 production may not be solely related to changes in PK-C activation and in [Ca++]i but may reside in other than transmembrane signalling mechanisms required for IL2 production.

Meningococcal antigens (MA) showed adjuvant activity when administered to mice at the same time as antigen (sheep erythrocyte (SE], by increasing the splenocyte plaque-forming response in a dose-related manner. However, when SE were given 1 day after MA administration, the subsequent plaque formation was diminished from normal in proportion to the dose of MA injected. Splenocytes taken from mice up to 5 days after MA injection actively inhibited plaque formation when mixed with splenocytes immunized with SE 4 days earlier. Two days after MA injection the nonspecific inhibition of plaque formation was mainly due to adherent spleen cells, while at 5 days nonadherent cells had acquired the inhibitory activity. It appears that it is the degree of activation of adherent cells resulting from the timing and dosage of MA which modulates the subsequent development and secretion of antibody-forming cells.

An extract of the meningococcus antigens (MA) prepared from N. meningitidis was tested for an anti-tumor effect in rat and murine metastasizing tumor models. Effectiveness of MA in each model varied with dose and was manifested as significantly improved survival of the treated animals. Growth of the primary Fischer bladder carcinoma (FBCa) and metastases to lungs and lymph nodes were significantly inhibited in F344 rats treated weekly with 1 mg MA. Administration of MA at 100 micrograms per animal significantly prolonged survival of P815 mastocytoma-inoculated DBA/2 mice. Survival of C-26 colon adenocarcinoma-bearing Balb/c mice was significantly improved in animals that received weekly injections of 20 micrograms MA, without significant effect on the development of local tumor. The meningococcal antigens demonstrate strong mitogenic activity in B-cell-enriched murine spleen cultures. Thus the immunostimulatory activity of MA in experimental malignancy could involve, directly or indirectly, activation of B lymphocytes.

This report examines the capacity of autologous and exogenous interleukin-2 (IL2) to regulate and/or induce immunoglobulin M (IgM) production in these patients. Pokeweed mitogen (PWM)-induced lymphocyte proliferation and PWM- and IL2-induced IgM secretion were monitored in vitro during the postburn period (10 to over 60 days) in 40 patients aged 16-72 years, with burns 20-90 per cent TBSA. PWM-induced IgM secretion fluctuated considerably during this period. Twelve of 40 patients demonstrated no IgM production and a significant (P less than 0.001-0.05) proportion of them had profoundly suppressed levels. Of the survivors, restoration of IgM secretion to normal levels was achieved in only 60 per cent at time of discharge. Even more consistently suppressed was exogenous IL2-driven production of IgM. In contrast, PWM-induced lymphoproliferation was normal in over 70 per cent of the patients. Thus, the T-cell-dependent antibody response was suppressed for long periods of time, possibly from some deficiency in IL2-regulated secretion or reception of helper T-cell-derived factors necessary for B cell differentiation into Ig-secreting cells.

The mouse immune response against Neisseria meningitidis was studied by using an extract from group Y (Slaterus) known to contain protein antigens common to other meningococci. By using a solid-phase radioimmunoassay, high titers of specific IgM and IgG class antibodies were measured which lasted over 2 months after immunization. These antibodies cross-reacted with similar extracts from other meningococci groups. Bactericidal antibodies directed against protein antigens were also elicited after immunization and they belonged to IgM, IgG2a, and IgG2b isotypes. Cellular immunity, expressed as delayed type hypersensitivity under the conditions tested, could be detected neither in homologous nor heterologous reactions.

Serum concentrations of receptor for the T cell growth factor interleukin 2 (IL-2R) were compared to the capacity of activated T cells to express surface IL-2R in patients with major burns. In immunosuppressed patients the numbers of cells expressing IL-2R were transiently (survivors) or permanently (nonsurvivors) reduced (up to 50 and 90%, respectively). In contrast, the levels of soluble IL-2R in patients' sera were significantly (P less than 0.001-0.05) elevated throughout the postburn period. Within 24 hr postinjury, over 90% of patients demonstrated 400-3500 U/ml of serum IL-2R compared with 120-340 U/ml in normal controls. Soluble IL-2R, when patients became immunosuppressed, further increased to 4445 +/- 962 U/ml in nonsurvivors and to 2031 +/- 578 U/ml in survivors, and demonstrated a significant capacity to inhibit the activity of exogenous IL-2. In survivors the levels of serum IL-2R declined at discharge, to 1009 +/- 104 U/ml. Thus, in thermally injured patients, soluble IL-2 receptor concentrations are elevated, and could interfere with IL-2-mediated immune interactions.

In the burn patient, the mechanisms leading to impaired T lymphocyte activity are unclear. The capacity for T cell proliferation and the expression of Tac antigen (IL-2 receptor) was assessed during the post-burn period in patients with injuries ranging from 5-68% total body surface area. T cell-dependent (polyclonal) immunoglobulin synthesis, mixed lymphocyte reaction and Interleukin-2 production were also determined in these patients and correlated with survival. Surviving patients demonstrated a transient reduction while terminal patients exhibited a permanent reduction in the number of Tac (+) lymphocytes, unrelated to the absolute number of T cells, during the post-burn period. The reduced percentage of IL-2 receptor-expressing T cells coincided with the suppressed antibody response and reduced alloreactivity. Although the concentration of IL-2 was decreased in all patients throughout the hospitalization period, surviving patients showed a gradual increase in its production while terminal patients gradually decreased to undetectable levels. Exogenous recombinant IL-2 induced a significant enhancement of in-vitro polyclonal immunoglobulin production and blastogenesis in the mixed lymphocyte reaction in immunosuppressed patients who demonstrated up to 50% reduction in the percentage of IL-2 receptor positive cells. Thus, the reduced capacity for production of and response to IL-2 after thermal injury may lead to the immunosuppression due to a lack of T lymphocyte clonal expansion. The permanent nature of this defect in patients who died from fatal sepsis may suggest a causative relationship.

The capability of exogenous recombinant Interleukin 2 (rIL2) to increase expression of the IL2 receptor (IL2R), and to augment the in vitro proliferative response of lymphocytes from immunosuppressed burned patients (5-80% full-thickness burns) was examined. Throughout the postburn period the percentage of IL2R-bearing cells in Concanavalin A-activated cultures of patients' peripheral blood mononuclear cells (PBMC) was measured by direct immunofluorescence with monoclonal anti-IL2R antibodies. Mitogen-induced IL2R expression was decreased by 40-90% in cultures of patients' PBMC, parallel to their reduced alloreactivity. During this period of immunosuppression supplementation of the mitogen-activated cultures with recombinant IL2 (20 U/ml) significantly increased the number of IL2R-expressing cells in all patients studied. However, IL2-induced enhancement of blastogenesis in the MLR was observed only with the patients who, in mitogen-activated cultures, sustained numbers of IL2R-endowed cells at least 50% of their baseline (the level within the first 24 hours postburn). Also, unstimulated PBMC of the same responding patients demonstrated a restoration of proliferation in the presence of rIL2. These patients were survivors. Thus the proliferative response correlated well with the number of Con A- but not rIL2-induced IL2R-expressing cells. These results suggest that in burned patients, IL2 up-regulates its receptors, but they may represent low-affinity nonfunctional receptors. Thus thermal injury appears to affect expression of functional (high-affinity) receptors.

A human whole blood chemiluminescence (CL) assay was established using zymosan as cell activator. Aroclor 1254 was found to inhibit this CL response in a direct linear relation to its concentration, (50% inhibitory dose, (ID50) equal to 5 x 10(-4)M) in diluted blood samples of 10 normal human subjects. In comparison the ID50 of other inhibitors was 1.3 x 10(-3)M for ethylenediamine tetraacetic acid, 3.3 x 10(-3)M for ascorbic acid, 4 x 10(-3)M for reduced glutathione, 1.2 x 10(-1)M for ethanol, 2.5 x 10(-1)M for methanol and 3.7 x 10(-1)M for dimethyl sulfoxide. Using 12-o-tetradecanoyl-phorbol-13-acetate (TPA) as cell activator the CL response was likewise inhibited by Aroclor 1254 with an ID50 of 4.5 x 10(-4)M. However, it was found that Aroclor 1254 alone has a stimulatory CL effect on otherwise unactivated cells. To compare the mechanisms involved in the CL elicited by the three stimulants zymosan, TPA and Aroclor 1254, the CL signal was measured in the presence of cytochalasin B. Cytochalasin B inhibited zymosan-induced CL, had a smaller inhibitory effect on TPA-induced CL but it could augment the CL response initiated by Aroclor 1254. This pattern of responses implicates Aroclor 1254 in the activation of eicosanoid metabolism as it matches the differential responses reported for arachidonic acid.

A preparation of meningococcal antigens (MA) extracted in CaCl2, and containing mostly outer membrane proteins, was strongly mitogenic for murine B lymphocytes. Given to mice in vitro, MA markedly impaired subsequent in vivo T-cell responses of splenocytes. Suppression of normal T splenocytes in vitro occurred with both adherent (Ad) and nonadherent (NA) splenocytes from MA-sensitized mice. B cells were much less affected by the suppression induced by MA, and only Ad cells could convey in vitro the low level impairment of B-cell proliferation. Strong T-cell suppression associated with a B-cell mitogen is also produced by bacillus Calmette-Guérin (BCG) and Corynebacterium parvum. The possible role of these phenomena in meningococcal disease is discussed.

The growth-inhibiting factor found earlier in human cell cultures appeared to be a ketoaldehyde. An infrared spectral study of 2,4-dinitrophenylhydrazine derivatives suggested that the ketoaldehyde was 4-hydroxy-2-ketobutyraldehyde. The compound, when synthesized, gave the same DNPH derivatives as the natural substance.

Used medium from cultured human cells contains a factor that inhibits growth of the "less virulent" strains of pathogenic bacteria, but only retards growth of the "more virulent" strains. The factor is heat-stable, dialyzable, and unaffected by change in pH; it chromatographs as material of molecular weight between 700 and 1500. There is evidence that this factor is an alpha-ketoaldehyde attached to a carrier.