Immunostimulatory principles from Chlorella pyrenoidosa--part 1: isolation and biological assessment in vitro.

Our proprietary preparation obtained by extraction of Chlorella pyrenoidosa cells, ONC-107 (Respondin), was recently found to selectively boost antibody response to the influenza vaccine in a human clinical trial. Respondin is a potent stimulator of mouse B cell proliferation and an activator of macrophages. Bioactivity-guided resolution concluded that Respondin is composed of a mixture of immunostimulatory principles of different chemical nature. A combination of size exclusion, anion exchange and hydrophobic interaction chromatography revealed that the bulk of the immunostimulatory activity resides in polysaccharide/protein complexes with molecular masses larger than 100 kDa that are composed primarily of galactose, rhamnose and arabinose.

Immunomodulating principles from shark cartilage. Part 1. Isolation and biological assessment in vitro.

Extracts from shark cartilage exhibiting powerful immunostimulating activity in vitro are described. The study shows that a simple extraction with water is very effective in producing the immunostimulating principles and implies that it is ideal for scale-up and manufacturing on a large scale. The extracts are potent stimulators of B cells and macrophages isolated from BALB/c mice spleen while stimulation of T cells was insignificant in our in vitro models. The study demonstrates that the active principles are thermally stable proteoglycans with molecular masses exceeding 100 kDa. This in vitro study represents an important step needed for further assessment of the products in vivo and their value for nutraceutical use.

Oral exposure to alloantigen generates intragraft CD8+ regulatory cells.

We have previously reported that oral administration of allogeneic rat spleen cells before kidney allotransplantation significantly prolongs graft survival. This prolongation was alloantigen specific and was associated with a decrease in graft-infiltrating cells (GIC) and an increase in transcription of IL-4 mRNA in the GIC. In this study increased splenic mixed lymphocyte responses from animals orally exposed to alloantigen before kidney transplantation suggested that the kidney allograft prolongation was not due to a masking of allorecognition, but to an immunomodulation of the immune response. We have assessed GIC T cell subsets on day 5 post-transplant and found decreased numbers of CD4(+) T cells in fed animals compared with controls, but there was no change in CD8(+) T cell numbers. The CD8(+) GIC from fed animals transcribed substantial levels of perforin, granzyme, and Fas ligand mRNA, indicating the presence of active CTL. Direct CTL assays showed that the GIC from fed recipients exhibited higher allo-CTL activity than GIC from control unfed recipients. In addition, the CD8(+) GIC exhibited high levels of IL-4 mRNA, suggesting Tc2-type regulatory cells. Prolonged graft survival in the face of active CTL and Tc2 cells suggests the presence of a CD8(+) regulatory cell population in the allograft. To confirm this, cell transfer experiments were performed. Prolongation of graft survival was transferred from rats orally exposed to alloantigen to naive animals by transfer of CD8(+) GIC. This is the first report that oral exposure to alloantigen prolongs kidney allograft survival by the generation of intragraft CD8(+) regulatory cells.

Prolongation of survival of primary renal allografts by feeding of donor spleen cells.

We have found that feeding Brown Norway (BN) rat spleen cells to Lewis rats prior to transplanting BN kidneys prolongs allograft survival (mean: 8.8 days in unfed rats, 21 days in the BN cell-fed rats; longest survival: 11 days without allo-feeding vs. 37 days with feeding). We have also found that feeding BN cells both before and after transplantation further extends survival (mean: 38 days; longest survival: 105 days). We also examined the cells infiltrating the grafts during the early stages of the allograft response (day 5). Using flow cytometry, we found a significant decrease in the number of leukocytes infiltrating the transplanted kidneys of fed animals. This decrease was mainly due to a drop in the number of infiltrating T cells. We also found that cytokine mRNA production by the graft-infiltrating lymphocytes, assessed by reverse transcription polymerase chain reaction, showed a significant increase in interleukin-4 and transforming-growth factor-beta mRNA in the graft-infiltrating lymphocytes of fed animals compared with the controls.

Immunostimulation of murine spleen cells by materials associated with bovine milk protein fractions.

Purified bovine milk proteins that were added to cultures of murine spleen cells significantly increased cell proliferation and production of immunoglobulin M. Casein and a whey mixture consisting of alpha-lactalbumin, bovine serum albumin, bovine gamma globulin, and beta-lactoglobulin (beta-LG) were stimulatory. Of the three beta-LG preparations that are commercially available (beta-LG containing variants A and B, purified variant A, and purified variant B), the unseparated mixture containing both the A and B variants showed the most immunomodulatory activity. Both alkaline treatment and trypsin digestion of the beta-LG preparation markedly reduced its effectiveness. Polymyxin B, while greatly diminishing the stimulatory effect of lipopolysaccharide, had no significant effect on either the enhancement of cell proliferation or the enhancement of immunoglobulin production by beta-LG. In the presence of S-(n-butyl)-homocysteine sulfoxamine, the stimulatory effect of beta-LG on cell proliferation and IgM production in vitro was markedly reduced.

A substrate amplification system for enzyme-linked immunoassays. II. Demonstration of its applicability for measuring anti-DNA antibodies.

We have recently described a substrate amplification system, based on the method of Self, which increases the sensitivity of alkaline phosphatase (AP)-dependent enzyme-linked immunosorbent assays (ELISA) by a factor of 30-50. This increase is achieved by having AP, the primary enzyme, produce an activator for a secondary enzyme-substrate system, within which marked amplification occurs. We have now demonstrated that this amplification method can be applied to the measurement of human antibodies to DNA. The sensitivity is greater by a factor of 10 than the conventional method, which uses p-nitrophenyl phosphate (p-NPP) as substrate. On replicate assays the method is reproducible, with a coefficient of variation of less than 0.1. This great increase in sensitivity should be of value in conserving specimens of serum and in screening monoclonal antibodies.

Lymphocytotoxic antibodies and intermediate immune complexes in hypergammaglobulinemic purpura of Waldenström.

Immune complexes, lymphopenia, and lymphocytotoxic antibodies were detected in two patients with hypergammaglobulinemic purpura of Waldenström. These abnormalities were further characterized in one patient. Ultracentrifugation analysis of this patient's serum showed an intermediate peak likely representing medium sized immune complexes. Circulating IgG-containing immune complexes (2-ME resistant Clq binding material) and IgM rheumatoid factor (2-ME sensitive) were also detected. A sequential study of cold reactive IgM lymphocytotoxic antibodies revealed a significant inverse correlation between antibody levels and peripheral lymphocyte counts. The possible implications of these findings in the pathogenesis of hypergammaglobulinemic purpura are discussed.

Pathogenesis of trehalose dimycolate-induced interstitial pneumonitis. IV. Evidence against roles for immunoglobulins and the complement system.

We showed previously that trehalose dimycolate (TDM) in oil administered intraperitoneally into susceptible mice produced interstitial and hemorrhagic pneumonitis by the seventh day after injection and that mature T cells are necessary for the production of these lesions. TDM has been reported to activate complement and to be chemotactic for macrophages in vitro. Accordingly, we looked for involvement of humoral mechanisms in the pathogenesis of TDM-induced pneumonitis. Genetically C5-deficient B10D2/oSn mice developed pulmonary lesions just as well as C5-sufficient mice. No activation of C3 occurred in the plasma of TDM-treated mice as determined by crossed immunoelectrophoresis. Some splitting of C3 occurred in bronchoalveolar lavage fluids, but this was similar in control and experimental mice. By immunofluorescence microscopy, there was no deposition of C3 or immunoglobulins (Ig) along the alveolar membranes. These findings and our published data provide additional evidence that TDM-induced interstitial inflammation in mice is exclusively a T-lymphocyte-dependent process.

The effects of lesions in the lateral septal and hippocampal areas on the humoral immune response of adult female rats.

There are interactions between the immune and the endocrine systems. A major role for adrenal steroids in the modulation of the immune system is well documented and the immune system can apparently activate the adrenal axis at the hypothalamic and/or pituitary level. Sex steroids modify immune function and sex differences in immune function suggest that there are also major interactions between the reproductive system and the immune system. To the extent that the endocrine system is integrated with the immune system, many of the brain areas and neural mechanisms implicated in psychoneuroendocrine processes may be involved in the regulation of psychoneuroimmune processes. We have proposed that the septal region represents an important extrahypothalamic and now propose that the septal region may also represent an important component in the central regulation of psychoneuroimmune processes. We have tested the effects of electrolytic and kainic acid (KA) lesions in the lateral septal area of female rats and have compared the effects of KA lesions in the lateral septal area with KA lesions in the hippocampal region on antibody production following immunization with 100 micrograms ovalbumin in complete Freund's adjuvant. Blood samples were collected on Days 0, 7, 14, 17, and 28 following immunization in the first experiment and on Days 0, 7, 14, and 28 in the second study. All animals were injected again with ovalbumin on Day 14. The IgG, IgA, and IgM antibody titers were then measured by an enzyme-amplified ELISA assay. KA lesions in the lateral septal area produced extensive cell loss in this region and produced a concurrent loss of cell bodies in the CA 3-CA 4 hippocampal areas. Compared to sham and electrolytic septal lesioned rats, KA lesions of the lateral septal area significantly reduced IgG titers on Days 7 and 17 with a trend for Day 14. Similarly, the IgA and IgM titers were significantly lower on Days 7, 14, and 17 for the rats with KA lesions in the lateral septal area. In addition, the KA septal lesioned rats gained more body weight and had higher thymus weights, relative to the electrolytic lesioned and sham groups. In addition, the rats with KA lesions in the hippocampal area showed significantly higher IgM titers on Day 7, elevated IgG titers on Day 14, and lower IgA titers on Day 28, relative to the sham group.(ABSTRACT TRUNCATED AT 250 WORDS)

Scleroderma and hemolytic anemia in a patient with deficiency of IgA and C4: a hitherto undescribed association.

We describe a 66-year-old women with CREST variant of scleroderma who developed autoimmune hemolytic anemia responsive to prednisone and demonstrated a deficiency of IgA and the C4 component of complement. This association of clinical, immunologic and genetic findings has not previously been reported. The literature relating to these findings is reviewed and possible mechanisms are discussed.

A substrate amplification system for enzyme-linked immunoassays. Demonstration of its general applicability to ELISA systems for detecting antibodies and immune complexes.

Self recently described a substrate system for alkaline phosphatase (AP)-dependent ELISAs which markedly increased sensitivity, compared to using p-nitrophenyl phosphate. This increase is achieved by having AP, the primary enzyme, produce an activator for a secondary enzyme-substrate system, within which marked amplification occurs. We adapted this technique to study antibodies to casein, bovine serum albumin, ovalbumin, and cardiolipin in the sera of patients with systemic lupus erythematosus (SLE) and normal individuals. The new substrate system yielded titres 30-50-fold higher than those with p-nitrophenyl phosphate (Sigma 104, p-NPP). In addition, when used in a solid-phase C1q binding assay we were able to use a 1 : 100,000 dilution of AP-conjugated anti-human IgG with the amplified substrate, compared to the 1 : 1000 dilution needed with p-NPP. This system is extremely valuable because of its flexibility. It can either be very sparing of limited samples, or if the added sensitivity is not needed, 100-fold less AP conjugate may be used. Thus rare or expensive conjugates can be significantly conserved.

Familial systemic lupus erythematosus: evidence for separate loci controlling C4 deficiency and formation of antibodies to DNA, nRNP, Ro and La.

A family has been identified in the Canadian province of Prince Edward Island in which 2 sisters have systemic lupus erythematosus in a sibship of 14. Studies are reported on 11 of the siblings and 16 other family members. The affected siblings, and 4 other members of their sibship, are halfnull homozygotes for the C4A component of complement. We studied the distribution in family members of antibodies to ss and dsDNA, and to Ro(SSA), La(SSB), Sm and nRNP. Eight of 11 members of the affected sibship are antibody producers, compared to only 3 of 13 members of the parental generation. Our study provides further evidence for an association between null genes for C4A and familial lupus, and suggests, in an unusually large kindred, that several other genetic factors are involved in the production of antinuclear antibodies.

Failure of oral tolerance in (NZB X NZW)F1 mice is antigen specific and appears to parallel antibody patterns in human systemic lupus erythematosus (SLE).

Primary oral antigen exposure normally induces mucosal immunity and an active suppression of the systemic immune response. Patients with systemic lupus erythematosus (SLE) have increased antibodies to bovine gamma-globulin (BGG), which suggested a possible failure of oral tolerance in SLE. We examined this possibility in murine lupus. NZB/W females were fed BGG or saline and were subsequently immunized ip. Primary and secondary responses were assessed. At 1 month of age the mice tolerized normally in response to feeding with BGG but, at 4 months of age, not only did they not tolerize, the mice fed BGG had a 5- to 7-fold higher response to parenteral immunization than did the saline-fed mice. Control strain mice tolerized normally at both ages (a 5- to 10-fold lower response). Conversely, when fed ovalbumin, NZB/W females tolerized normally at both 1 and 4 months of age, and patients with SLE had normal levels of antibody to this antigen. However, we also found increased levels of antibodies to bovine casein in SLE patients, and found that NZB/W mice failed to orally tolerize with this antigen at either 1 or 4 months of age. Thus, the failure of oral tolerance in the NZB/W mice appears to be antigen specific and age dependent and, at least with respect to these three antigens, appears to parallel the antibody patterns seen in human SLE.

Dietary bovine antigens and immune complex formation after intravenous immunoglobulin in common varied immunodeficiency.

Previously we have shown that the sera of hypogammaglobulinemic patients may contain large amounts of antigenically intact foreign protein of dietary origin, presumably due to the absence of an adequate gastrointestinal secretory immune barrier. In these studies we show that the intravenous infusion of immunoglobulin in these patients may result in high levels of immune complexes postinfusion and that at least one constituent of these complexes is likely to be bovine casein.

Orally induced tolerance to casein in mice on normal mouse chow.

We examined the ability of bovine casein to induce oral tolerance in BDF1 mice raised on a normal mouse chow diet. Giving 20 mg casein 1 X or 4 X by intragastric tube or for 28 days at 1 mg/ml in drinking water failed to have a significant effect (P greater than 0.2) on the subsequent immune response to parenteral casein, suggesting that casein was not an effective oral tolerogen. However, we discovered that most normal mouse chow contains casein. When BDF1 mice were raised on a casein-free diet, and treated as above, they showed marked suppression of subsequent responses to parenteral casein (P less than 0.01). Our results indicated that mice on the normal diet were presuppressed by the dietary exposure. Examination of several other normal mouse chows revealed that they all contained casein as a protein source. A possible effect of prior exposure must be considered in all experimental animal studies involving immune responses to antigens which may be present in the environment.

Dietary protein antigenemia in humoral immunodeficiency. Correlation with splenomegaly.

Enhanced gastrointestinal absorption of dietary substances is an important feature of normal neonatal life that also exists in particular disease states such as selective IgA deficiency and atopic allergy. In these studies, it is shown that patients with hypogammaglobulinemia have increased absorption of dietary bovine antigens and that most patients have large amounts of these proteins present in the serum even after an overnight fast. The amounts of such proteins were found to be correlated with spleen size and/or peripheral lymphoid hypertrophy. Interestingly, three patients with X-linked agammaglobulinemia did not have detectable amounts of these proteins in the serum nor did they have splenomegaly or lymphadenopathy. It is speculated that hypogammaglobulinemic patients have a specific gastrointestinal mucosal lesion that permits the chronic excessive absorption of dietary antigens and may result in lymphoid hypertrophy.