Cytokine-inducing activity of a proline-rich polypeptide complex (PRP) from ovine colostrum and its active nonapeptide fragment analogs.

A complex of proline-rich polypeptides (PRP) was isolated from ovine colostrum in our laboratory and was shown to possess immunomodulatory properties and psychotropic activity, including beneficial effects in the treatment of Alzheimer's disease. A nonapeptide fragment (NP): Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro, isolated from the chymotryptic digestion products of PRP, and its C-terminal fragment, a hexapeptide (HP): Tyr-Val-Pro-Leu-Phe-Pro also exhibited immunoregulatory activity. Although NP and HP expressed activity similar to that of PRP in studies on humoral and cellular immune responses, in other immune processes, e.g. induction of cytokines, they showed markedly lower activity than PRP. In the search for more active peptides, in the present study, we compared the cytokine-inducing ability of PRP, NP, HP, and linear oligomers of NP or HP. For this purpose, the induction of IFN, TNF-alpha, IL-6, and IL-10 in human whole blood cell cultures was measured. NP, HP, and their oligomers showed differential effects in the induction of cytokines, generally lower than that of PRP. Only the PRP complex showed a bell-shaped dose-response dependence suggesting regulatory properties. There were no distinct differences between monomeric forms of NP (NP1) or HP (HP1) and their oligomers in the induction of IFN and TNF-alpha (Th1 cytokines) but such differences were found in the induction of IL-6 and IL-10 (Th2 cytokines). Dimer (NP2) was less active than the monomeric NP1 nonapeptide in the induction of IL-6 and IL-10. On the other hand, oligomers: HP3 and HP4, showed a significantly higher ability to induce Th2 cytokines compared to HP1, HP2 or NP peptides. This was especially evident in the case of IL-10 induction, where the activity of HP4 surpassed the activity of PRP and approached the activity of LPS-PHA. The results obtained showed that some of the peptides studied, when used at higher concentrations (100 microg/ml) may replace the PRP complex as cytokine inducers. Our data also suggest the possibility of using certain oligomers for selective induction of particular cytokines.

Colostrinin: a proline-rich polypeptide (PRP) complex isolated from ovine colostrum for treatment of Alzheimer's disease. A double-blind, placebo-controlled study.

A proline-rich polypeptide (PRP) complex, subsequently called Colostrinin, was isolated from ovine colostrum. The complex showed immunomodulatory properties in mice, rats, and chickens, inducing maturation and differentiation of thymocytes. It was recently found that Colostrinin is a cytokine-like factor that acts as an inducer of interferon gamma (IFN-gamma) and other cytokines in human peripheral blood and cord blood leukocyte cultures and has psycho-immuno-enhancing activity in volunteers. These observations prompted us to study the effect of Colostrinin on patients with Alzheimer's disease (AD). Forty six AD patients were divided into 3 groups and randomly assigned to receive orally either Colostrinin (100 microg per tablet, every second day), commercially available bioorganic selenium (100 microg selenium per tablet, every second day) or placebo tablets. One cycle of the treatment lasted 3 weeks and was separated from the next cycle by a 2 week hiatus. Each patient received 10 cycles of treatment during the year of the clinical trial. Outcomes were assessed by psychiatrists blinded to the treatment assignment. Eight of the 15 AD patients treated with Colostrinin improved and in the 7 others the disease had stabilized. In contrast, none of the 31 patients from the selenium or placebo groups with similar mild or moderate AD improved. The administration of selenium promoted stabilization in 13 of the 15 patients, whereas in the placebo group only 8 of the 16 patients were stabilized at the 12 month trials end-evaluation. Colostrinin was found to be a remarkably safe drug. Mild and transient effects were anxiety, stimulation, insomnia, and tiredness. The results obtained showed that oral administration of Colostrinin improves the outcome of AD patients with mild to moderate dementia. The results are very encouraging and deserve further research.

Colostrinin, a polypeptide isolated from early milk, facilitates learning and memory in rats.

Initial observations in humans indicated that colostrinin, a complex of polypeptides derived from the colostrum of sheep, facilitates cognitive functioning in patients with Alzheimer's disease. Its effect on learning and memory in more controlled settings as well as the specificity of these effects were, however, unknown. The present experiments evaluated the effects of colostrinin on spatial learning (Morris water maze) and incidental memory (habituation test) in male Wistar rats of two age groups. Colostrinin, at a dose of 4 microg/rat IP, facilitated acquisition of spatial learning of 13- (aged) but not 3-month-old (young) rats. At the same dose, it improved incidental learning in aged rats, while the dose of 20 microg/rat attenuated it. Colostrinin did not change locomotor activity of rats. Taken together, the present findings indicate that colostrinin may have some beneficial effects on cognitive functioning, particularly in aged subjects. Given the fact that colostrum is the first nutritive agent of neonates, it might be speculated that its peptides may facilitate the early postnatal development of the cerebral neurons and their plasticity.

Effect of colostrinin, an immunomodulatory proline-rich polypeptide from ovine colostrum, on sialidase and beta-galactosidase activities in murine thymocytes.

Colostrinin: a proline-rich polypeptide (PRP) from ovine colostrum and its nonapeptide active fragment (NP) induce maturation and differentiation of murine thymocytes, formation of helper cells from PNAhigh thymocytes and cytotoxic T cells from PNAlow thymocytes. These processes are accompanied by changes in expression of receptors for peanut agglutinin (PNA), PNAhigh thymocytes were transformed into PNAlow cells, and vice versa. It was shown, in various laboratories, that sialyltransferases are involved in the transformation of PNAhigh thymocytes into PNAlow cells. To find out whether the expression of receptors for PNA on murine thymocytes might also be influenced by other enzymes, we decided to study the effect of PRP and NP on sialidase and beta-galactosidase activities in these cells. The results obtained showed that the most of sialidase activity of murine thymocytes is present in the plasma membrane compartments. Both thymocyte subpopulations PNAhigh and PNAlow, showed similar sialidase activity, which was not affected either by PRP or NP. In contrast to sialidases, most of beta-galactosidase activity was present in the cytosol. PNAhigh, thymocytes showed a higher beta-galactosidase activity than PNAlow cells. Incubation of immature, PNAhigh, thymocytes with PRP or NP enhanced the beta-galactosidase activity in these cells. The presented results suggest that sialidases seem not to be involved in modulation of surface sialic acid content during murine thymocyte maturation. On the other hand, stimulation of activity of beta-galactosidase in PNAhigh, immature thymocytes by PRP and NP suggests that beta-galactosidase in murine thymocytes might be involved in transformation of PNAhigh into PNAlow cells.

Coincidence between spontaneous release of interferon and tumor necrosis factor by colostral leukocytes and the production of a colostrinine by human mammary gland after normal delivery.

We have tentatively identified colostrinines as novel cytokines produced by the mammary gland after delivery and detectable in colostrum. The primary colostrinine, the proline-rich polypeptide, was isolated from ovine colostrum in 1974. It is generally understood that the various factors present in colostrum play a pivotal role in transmitting of passive or active immunity from mother to child. We have found previously that both ovine and human colostrinines are inducers of interferon (IFN) gamma and other cytokines. In this paper, we reported that the leukocytes isolated from human colostrum donated by healthy mothers at 1-9 days after delivery, produced IFNs and tumor necrosis factors (TFNs) spontaneously. The release of IFNs and TNFs coincided with production of a colostrinine that has been isolated from the human colostrum samples and partially characterized. Our results suggest that the maximum production of colostrinine occurs 3 days after delivery. The tolerance (hyporeactivity) of the colostral leukocytes to IFN inducers and the modulation of the TNF response may be the late effects of the colostrinine release.

Colostrinine: a proline-rich polypeptide from ovine colostrum is a modest cytokine inducer in human leukocytes.

A proline-rich polypeptide (PRP), now named colostrinine, molecular weight 18,000, was isolated from ovine colostrum and characterized by Janusz, Lisowski et al. The nonapeptide (NP) which is an active fragment of PRP was obtained by chemical synthesis. In mice, PRP has many regulatory effects on the humoral and cellular immune response. The present paper describes PRP as a cytokine inducer. PRP at concentration of 1-100 micrograms/ml was found to induce production of interferon (IFN) and tumor necrosis factor (TNF) in human peripheral blood leukocytes and in whole blood cultures. The effects were dose related. The identified till now cytokines induced by PRP were IFN-gamma and TNF-alpha but many other cytokines may be stimulated also. NP was considerably less active as the cytokine inducer than the natural PRP. Two volunteers given orally once daily for two to three weeks 100 or 200 micrograms PRP in tablets were found to develop the tolerance of IFN induction and had the modified TNF response. Furthermore, the PRP-treated volunteers showed signs of psycho-stimulation. Taken together our observations suggest that ovine PRP is active in humans and may have therapeutic value as an immunostimulant and/or neurotropic cytokine.

Proline-rich polypeptide (PRP)--an immunomodulatory peptide from ovine colostrum.

The structure and properties of a new immunomodulatory peptide isolated from ovine colostrum are described. PRP acts both in vivo and in vitro, and is not species specific. PRP increases permeability of skin vessels, and causes differentiation of murine thymocytes into functionally active T cells. It can simultaneously change surface markers and function of cells. The polypeptide is able to reduce binding of peanut agglutinin (PNA) to PNA+ thymocytes and to increase the binding of PNA to PNA- cells. PRP is also able to transform cortisone-resistant thymocytes into cortisone-sensitive, and vice versa. The observed changes occurred concomitantly, i.e. changes in binding of PNA were accompanied by changes in resistance to cortisone and in expression of helper or suppressor activity. The fact that changes induced by PRP are reversible after the second exposure of the cells to the polypeptide makes it unique among known immunomodulators. An active nonapeptide fragment: Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro was isolated from the products of PRP digestion. It shows full spectrum of biological activities of PRP. The sequence-Pro-Leu-Phe- is responsible for the immunological effect of the peptide.

A colostral protein that induces the growth and differentiation of resting B lymphocytes.

We describe the first protein of mammalian origin that induces the growth and differentiation of resting B lymphocytes. A proline-rich protein has been isolated from sheep colostrum. A purified proline-rich protein preparation (PRPP) induced resting mouse B cells into and supported their progression through the cell cycle at frequencies comparable with those seen for LPS. Differentiation of resting B cells to plaque formation was also supported as efficiently by PRPP as it was by LPS. However, PRPP was distinct from LPS in that it supported the growth and differentiation of resting B cells derived from either C3H/Tif or C3H/HeJ mice. Splenocytes from neonatal mice responded robustly to PRPP with the growth and differentiation of contained B cells to plaque formation. Unlike LPS, PRPP did not induce detectable Ig isotype switching.

Proline-rich polypeptide (PRP) from ovine colostrum. Bi-directional modulation of binding of peanut agglutinin, resistance to hydrocortisone, and helper activity in murine thymocytes.

A proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. The present report demonstrates that the polypeptide can cause bi-directional modulation of surface markers and function of murine thymocytes. PRP is able to reduce binding of peanut agglutinin (PNA) to murine PNA+ thymocytes and to increase the binding of PNA to PNA- cells. This transition of binding ability can be reversed by a second treatment of cells with PRP. PRP is also able to transform cortisone-resistant thymocytes into cortisone-sensitive, and vice versa. Helper cells induced by PRP from murine glass-nonadherent thymocytes (PNA+) showed suppressor activity after the second treatment with PRP. The observed changes were occurring concomitantly, i.e. changes in binding of PNA were accompanied by changes in resistance to cortisone and in expression of helper or suppressor activity. Bi-directional effects of PRP on PNA-binding ability, sensitivity to hydrocortisone, and helper-suppressor function, makes this polypeptide unique among immuno-modulators known.

Immunoregulatory properties of synthetic peptides, fragments of a proline-rich polypeptide (PRP) from ovine colostrum.

It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. A nonapeptide fragment Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro was isolated from the chymotryptic digest of PRP. The nonapeptide showed biological activity similar to PRP. The determined amino acid sequence was now confirmed by synthesis. Synthetic nonapeptide as well as its C-terminal hexapeptide, Tyr-Val-Pro-Leu-Phe-Pro, showed biological activity similar to PRP and the nonapeptide obtained from PRP.

Immunoglobulins of colostrum--VI. Comparative studies of cytophilic properties of bovine serum and colostral IgG.

In our previous studies, using physical-chemical and serological methods, substantial differences between bovine serum and colostral IgG, especially IgG2, have been shown. The structural differences were localized in the Fc region of immunoglobulins studied. The present comparative studies were undertaken to determine whether structural differences in the Fc region of bovine serum and colostral IgG are reflected in the interaction of these immunoglobulins with the guinea-pig peritoneal macrophage Fc gamma receptor. It was found that binding of bovine serum and colostral IgG1 was a saturable process and only quantitative differences in the mode of binding to the Fc receptor were observed. There is, however, a big difference in cytophilic activity of bovine IgG2--no saturable and reversible binding is observed in the case of bovine serum IgG2.

A proline-rich polypeptide (PRP) with immunoregulatory properties isolated from ovine colostrum. Murine thymocytes have on their surface a receptor specific for PRP.

A proline-rich polypeptide (PRP) was isolated from ovine colostrum. The polypeptide exhibits immunoregulatory properties. It induces maturation of murine thymocytes into functionally mature helper or suppressor T cells. To elucidate the mechanism of interaction of the polypeptide with thymocytes, effect of PRP immobilized on insoluble carriers on thymocytes was compared with the effect of soluble PRP. It was found that PRP covalently linked to cellulose discs or AffiGel 702 or adsorbed on polystyrene latex beads shows activity similar to activity of PRP in solution. A contact of PRP with the cell surface is satisfactory to induce maturation of thymocytes. PRP adsorbed on polystyrene latex beads forms rosettes with thymocytes. Formation of rosettes is specifically inhibited by soluble PRP. The results obtained suggest that murine thymocytes have on their surface a receptor specific for PRP.

Immunologically active nonapeptide fragment of a proline-rich polypeptide from ovine colostrum: amino acid sequence and immunoregulatory properties.

It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. To study the relationship between the structure of PRP and its immunomodulatory properties, the polypeptide was digested by chymotrypsin. Products of the proteolysis were separated by gel filtration and three fractions were obtained: PRP-1, PRP-2 and PRP-3. The activity of the fractions was compared with the activity of the untreated PRP. It was found that PRP-1 was inactive, whereas PRP-2 and PRP-3 showed an activity in the regulation of the immune response assayed by measurement of PFC, and by studying effects on delayed hypersensitivity, formation of autologous rosette-forming cell, and sensitivity of thymocytes to hydrocortisone. The activity of PRP-2 and PRP-3 was comparable to the activity of PRP. The PRP-3 fraction of low mol. wt was further purified and a pure nonapeptide of mol. wt 1000 (PRP-3b) was isolated. The amino acid sequence of PRP-3b was: Val--Glu--Ser--Tyr--Val--Pro--Leu--Phe--Pro. The nonapeptide showed the full spectrum of biological activities of PRP. Comparison of terminal amino acid suggested that PRP-3b was neither the NH2- nor the COOH-terminal fragment of PRP. The amino acid sequence of the nonapeptide indicated that PRP-3b is different from other known immunomodulators.

Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum.

A proline-rich polypeptide isolated from sheep colostrum is described. The molecular weight of the polypeptide determined by gel filtration is 17 200. However, in the presence of guanidinium chloride the molecular weight found is about 6000. The polypeptide contains about 22% of proline, a high proportion of non-polar amino acids, a low percentage of glycine, and no alanine, arginine and cysteine residues. The only N-terminal amino acid found is leucine. C.d. spectra in water and in 50% (v/v) trifluoroethanol suggest the presence of block sequences of proline residues forming helices of polyproline II type. The proline-rich polypeptide is soluble at 4 degrees C but is reversibly precipitated on warming to room temperature. Maximal precipitation is observed at pH 4.6 and at ionic strength above 0.6. The precipitation depends on the concentration of the polypeptide. No effect of other proteins, Ca2+ and Zn2+ ions on the precipitation of the polypeptide was found. The proline-rich polypeptide is not an amphipathic protein. The lack of effect of the polypeptide on proteolytic enzymes ruled out the possibility that it is an inhibitor of proteinases.

Immunoglobulins of colostrum. V. Localization of structural differences between serum and colostral ovine and bovine IgG1 and IgG2 immunoglobulins.

Studies on localization of structural differences between ovine and bovine serum and colostral IgG immunoglobulins are described. Comparison of heterogeneity, susceptibility to proteolytic enzymes, peptide maps, amino acid compositions, and antigenic properties of immunoglobulins and their Fab and Fc fragments and H and L chains showed that structural differences are localized in the Fc region. The strongest differences were found in case of IgG2. It was also shown that no Fc fragments could be obtained from bovine serm IgG2 and ovine serum and colostral IgG2 due to their susceptibility to papain and trypsin. The results obtained confirmed our suggestion that colostral IgG2 are locally synthesized in mammary glands, whereas colostral IgG1 might be a mixed population of molecules locally synthesized and transferred from the serum.

Proline-rich polypeptide from ovine colostrum: its effect on skin permeability and on the immune response.

The effect of a proline-rich polypeptide from ovine colostrum on the humoral immune response in mice against sheep red blood cells (SRBC) was studied. It was shown that the polypeptide had a regulatory activity stimulating or suppressing the immune response. The proline-rich polypeptide also increased the permeability of skin vessels in guinea-pigs. Studies on the mechanism of the effect of the proline-rich polypeptide suggest that prostaglandins are involved in the activity of the polypeptide.

Immunoglobulins of colostrum. V. Comparative studies of ovine and bovine serum and colostral IgM.

Comparative studies of physiocochemical properties of ovine and bovine serum and colostral IgM are described. Serologic properties, molecular weight, amino acid composition, carbohydrate content, peptide maps, heterogeneity, and ORD and CD spectra were compared. Colostral IgM have additional antigenic determinants, higher carbohydrate content, lower heterogeneity, and higher content of beta-structure. Different amino acid composition and different peptide maps of serum and colostral IgM were shown. The results obtained suggest that ovine and bovine colostral IgM immunoglobulins may be a mixed population of molecules locally synthesized and transferred from the serum.

Localization of structural differences between serum and colostral ovine and bovine IgG1 and IgG2 immunoglobulins.

Physicochemical and serological studies on immunoglobulins, their Fab and Fc fragments, L and H chains, showed that the strongest differences occurred in case of IgG2, and that the differences were localized in the Fc region.

Immunological activity of a proline-rich polypeptide from ovine colostrum.

The results presented in the paper showed that PRP increased permeability of skin vessels in guinea pigs and that PRP stimulates or suppresses the immunological response in mice, depending on the magnitude of the response.

Anti-TNP antibodies from bovine serum and colostrum.

Specifically purified anti-TNP antibodies of subclass IgGI and IgG2 were isolated using immunoadsorbent prepared from AH-Sepharose and TNP-BSA. Isoelectric focusing in poliacrylamide gel showed differences between antibodies obtained from colostrum and serum. These differences were more observable when H chains of IgG1 subclasses were compared. No differences were observed in L chains. The antibodies obtained were the non-precipitating ones. Spectral measurements in the presence of SO3- ions, showed that the antibodies studied were of low affinity (below 10(-6) M). The difference spectra showed, that binding of a hapten (epsilon-TNP aminocapronate acid) to the investigated antibodies caused the shift of the hapten's absorption bands indicating that the binding occured in the hydrophobic pocket of the antibody binding site. The strongest effect was observed in the case of antibodies of IgG1 subclass.