Potential therapeutic applications of PHA-L4, the mitogenic isolectin of phytohemagglutinin

Assuming that the attributes of the mitogenic-leukoaggglutinating (L4) isolectin of phytohemagglutinin as a proposed ideal biologic response modifier can be confirmed, it could prove to be a highly versatile agent with broad therapeutic potential for several areas of management including cancer and cancer surgery adjuvant, critical infections (including that with the human immunodeficiency viruses), vaccine adjuvant, allograft transplantations, aplastic anemias, and extensive burns. The isolectin is predictably more likely to be effective as an adjuvant or adjunctive agent than as an induction agent. Initial evaluation in dogs would serve the double purpose of establishing a presumptive key role in veterinary medicine and expediting the development of its use in humans.     

Characteristics of PHA-L4, the mitogenic isolectin of phytohemagglutinin, as an ideal biologic response modifier

Phytohemagglutinin retains the properties of a theoretically ideal biologic response modifier in that it is available in a purely mitogenic L4 isolectin form that is stable; previously studied extensively; applicable as a simple skin test to assess immune competence and guide therapy; broadly immunostimulating with respect to both activation and proliferation of effector cell pathways; amenable to targeting maneuvers; stimulative of endogenous cytokine production; conveniently administrable by multiple routes; applicable to both active and adoptive immunotherapies; rapidly interacting irreversibly with lymphocytes; readily applied as a vaccine adjuvant; apparently nonsensitizing; relatively nontoxic, with maximum effective levels well below those for major toxicity; free from stress induction; nononcogenic; noninfectious; related to other mitogenic lectins that have augmenting therapeutic potential; compatible with other therapeutic modalities and conductive to collaborative use of other BRMs; well-suited to application as a surgical adjuvant and for prophylaxis against malignancies or infections in susceptible individuals; applicative to debilitated, immunosuppressed, and myelosuppressed patients; probably compatible with pregnancy; and potentially cost-effective.     

Physicochemical properties of the human lymphocyte mitogenic factor induced by phytohemagglutinin]

Some physical and chemical properties of the mitogenic factor (MF) produced in vitro by PHA-stimulated human lymphocytes were investigated. Treatment of the culture medium with proteases proved to decrease the MF activity sharply. The MF was found to have an elution pattern in Sephadex and Bio-gel gels similar to proteins with the molecular weight of 20 000--30 000 daltons (peak--25 000). In experiments with the MF fractionation by disc-electrophoresis on 5% polyacylamide gel the MF formed a few discrete peaks in the fractions corresponding to the mobility of alpha1- and alpha2-globulins and transferrin. In isoelectric focusing the MF formed 3 fractions in the pH range of 4.5--8.3. Functional heterogeneity of the MF is suggested.     

Facile preparation of the alpha-Gal-recognizing Griffonia simplicifolia I-B4 isolectin

The B4 isolectin from Griffonia simplicifolia is of great utility as a reagent for the identification of alpha-D-galactopyranosyl end groups. Its separation from isolectins containing A subunits has been greatly improved by a simple, rapid procedure using a column of N-acetylgalactosamine coupled to vinyl sulfone-activated Sepharose 4B to selectively retain the A subunit-containing isolectins. The procedure has the advantages over previous affinity procedures of speed (the isolation of B4 isolectin can be achieved in one day), simplicity, and high degree of resolution of the B4 isolectin.     

Some properties of the mitogenic factor induced in human lymphocytes under the influence of phytohemagglutinin]

Production of the PHA-induced human lymphocyte mitogenic factor (MF) previously described by the author and its influence on the lymphoid cells were investigated. The PHA-antiserum and immunosorbents were used for the PHA inactivation. The PHA-stimulated lymphocytes produced the mitogenic factor (MF) during the G1 period and the beginning of the S period of the cell cycle. When the cells were cultivated in the protein-free medium, the level of the DNA and the protein synthesis was significantly decreased, but the level of the MF production was 1.6 times greater than that of the MF production in the protein-containing medium. Kinetics of the lymphocyte reaction to the MF is described: DNA synthesis (as judged by the H3-thymidine uptake) began on the 5th--6th day and reached the maximum on the 6th--7th day.     

Bacterial endotoxins: Comparison of mitogenic,polyclonal, antibody-inducing and toxicity activities

The mitogenic effects on mouse spleen lymphocytes were determined in a large series of commercially available and laboratory-prepared lipopolysaccharides (LPS) obtained fromEscherichia, Salmonella, Serratia andShigella species; part of these LPS preparations was chemically modified prior to testing. In order to establish whether the degree of mitogenic activity corresponds with other biological effects of these preparations, polyclonal activity, capability to induce specific antibody formation and toxicity were determined for selected LPS's with different mitogenic effects. Some of the detoxication procedures used succeeded in reducing the toxicity of LPS while preserving its high mitogenic activitione of the Fe-detoxified preparations of LPS (from the R-form ofShigella dysenteriae serovar 1) exhibited a medium-degree efficacy in all parameters studied. Generally, there was no correlation between the degree of mitogenic activity and the polyclonal and antibody-inducing activities, but in some instances polyclonal activity did correlate with the antibody-inducing activity.     

Relative mitogenic activities of various estrogens and antiestrogens

The abilities of a variety of estrogens and antiestrogens to stimulate DNA synthesis in the prepuberal rat uterus were compared. One microgram of each compound was administered in vivo via a single intraperitoneal injection. DNA synthesis was assayed in vitro in isolated nuclei 24 h later. The relative mitogenicities of the steroidal estrogens were: 16 alpha-E2 less than 17 alpha-E2 = E3 = 16-EpiE3 less than 16 beta-E2 = 17 beta-E2. The potencies of several nonsteroidal estrogens were also tested. Indenestrol A was as potent at 17 beta-E2, whereas indanestrol and dimethylstilbestrol had weaker activities. The antiestrogens, nafoxidine and 4-hydroxytamoxifen, were both potent stimulators of DNA synthesis. The abilities of an estrogen to stimulate increases in uterine wet weight, DNA polymerase alpha activities, and DNA synthesis in uterine nuclei 24 h after injection were closely correlated. Because the magnitude of the stimulation of DNA synthesis was greatest, its measurement is the most sensitive of these assays of uterotrophic activity.     

Structure and mitogenic activities of corn cob heteroxylans

Various structurally different water-soluble (ws) and water-insoluble (wis) heteroxylans have been tested for mitogenic and co-mitogenic activity and shown to differ in their stimulating potency. The ws arabinoglucuronoxylan (ws-AGX) from corn cobs exhibited the highest potency which was comparable to that of the immunomodulator zymosan, whereas the wis-AGX from corn cobs was inactive. Water-soluble derivatives of wis-AGX as well as hydrolytically modified ws-AGX fractions were prepared and tested for their mitogenic and co-mitogenic activity. The results indicate that the disaccharide side chains present only in ws-AGX might be important for expression of the immunostimulatory activity of this xylan.     

Further characterization of the combining sites of Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A(4).

Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A(4)(GS I-A(4)), which is cytotoxic to the human colon cancer cell lines, is one of two lectin families derived from its seed extract. It contains only a homo-oligomer of subunit A, and is most specific for GalNAcalpha1-->. In order to elucidate the GS I-A(4)-glycoconjugate interactions in greater detail, the combining site of this lectin was further characterized by enzyme linked lectino-sorbent assay (ELLSA) and by inhibition of lectin-glycoprotein interactions. This study has demonstrated that the Tn-containing glycoproteins tested, consisting of mammalian salivary glycoproteins (armadillo, asialo-hamster sublingual, asialo-ovine, -bovine, and -porcine submandibular), are bound strongly by GS I-A(4.)Among monovalent inhibitors so far tested, p-NO2-phenylalphaGalNAc is the most potent, suggesting that hydrophobic forces are important in the interaction of this lectin. GS I-A(4)is able to accommodate the monosaccharide GalNAc at the nonreducing end of oligosaccharides. This suggests that the combining site of the lectin is a shallow cavity. Among oligosaccharides and monosaccharides tested as inhibitors of the binding of GS I-A(4), the hierarchy of potencies are: GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc (Forssman pentasaccharide) > GalNAcalpha1-->3(LFucalpha1-->2)Gal (blood group A)()> GalNAc > Galalpha1-->4Gal > Galalpha1-->3Gal (blood group B-like)> Gal.     

An agglutinin with mitogenic and antiproliferative activities from the mushroom Flammulina velutipes

A hemagglutinin with a molecular mass of 12 kDa was isolated from the fruiting bodies of the mushroom Flammulina velutipes. Its molecular mass is similar to that of the fungal immunomodulatory protein isolated from F. velutipes (FIP-fve) with ice-cold 5% acetic acid and 50 mM 2-mercaptoethanol as extraction medium and to that of the larger 12 kDa subunit of F. velutipes lectin isolated with phosphate buffer as extraction medium. Its hemagglutinating activity cannot be inhibited by a variety of carbohydrates tested. The activity is stable between pH 4 and pH 11. Loss in activity occurred when the temperature is raised to 60 C and 70 C. Activity is indiscernible at and above 80 C. Its N-terminal sequence shows differences from that of FIP-fve. F. velutipes hemagglutinin stimulates [3H-methyl] thymidine uptake by mouse splenocytes. It inhibits proliferation of leukemia L1210 cells with an IC50 of 13 microM.     

Potent mitogenic activity of 4-acetylaminofluorene to the rat liver

Administration of 4-acetylaminofluorene (4AAF) to rats by oral gavage (1000 mg/kg) produces a wave of S-phase activity in the liver 36 h later, followed by a wave of mitoses at 48 h. These events were monitored by autoradiography of isolated hepatocytes and by histopathology, respectively. DNA-labelling was shown to occur following both in vivo and in vitro radiolabelling. The level of S-phases observed approached that reported following partial hepatectomy. These effects were not accompanied by unscheduled DNA synthesis (UDS) nor was any frank histopathological damage to the liver evident. 2-Acetylaminofluorene (2AAF) elicited a very weak S-phase response at a dose level of 50 mg/kg, but gave marked UDS between 12 and 48 h.     

Binding of Griffonia simplicifolia 1 isolectin B4 (GS1 B4) to alpha-galactose antigens

Glycoconjugates with terminal Galalpha1-3Galbeta1-4GlcNAc sequences (alpha-galactosyl epitopes, natural xenoreactive antigens) are present on various tissues in pigs and are recognized by human anti-alphagalactosyl (alphaGal) antibodies1. Hence xenotransplantation (pig-to-human) would trigger immune reactions involving complement activation and lead to the hyperactute rejection of the graft. Xenoreactive antigens are often studied by using the lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4), which shows high affinity to galactose. We here estimate the specificity of GS1 B4 for detecting various galactosyl epitopes by measuring lectin binding to neoglycoproteins, thyroglobulin and pig skeletal muscle. Enzyme linked lectin assays confirmed that GS1 B4 was highly specific to alpha-galactosylated neoglycoproteins while the lectin did not detect a beta-galactosylated ligand. The length of the sugar chains influenced the lectin-carbohydrate interaction. A monosaccharide linked to serum albumin showed higher lectin affinity than did neoglycoproteins with di- and tri-alpha-galactosyl epitopes. When the carbohydrate was extended, as in the xenoreactive pentasaccharide (Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), the carbohydrate- lectin interaction was meagre. Not only the terminal, but also the subterminal sugar affected the lectin binding because the GS1 B4 affinity to Galalpha1-3Gal was much stronger than to Galalpha1-3GalNAc. In bovine and porcine thyroglobulin most alphaGal epitopes appear to be cryptic, but are unmasked by a heat denaturation. In pig skeletal muscle there was lectin reaction not only in the muscle capillaries, but also in the connective tissue and intracellularly in muscle fibres. In Western blots of isolated proteins from pig muscle at least three bands were strongly stained after incubation with lectin.     

Identification of versican as an isolectin B4-binding glycoprotein from mammalian spinal cord tissue

Nociceptors are specialized nerve fibers that transmit noxious pain stimuli to the dorsal horn of the spinal cord. A subset of nociceptors, the nonpeptidergic C-fibers, is characterized by its reactivity for the plant isolectin B4 (IB4) from Griffonia simplicifolia. The molecular nature of the IB4-reactive glycoconjugate, although used as a neuroanatomical marker for more than a decade, has remained unknown. We here present data which strongly suggest that a splice variant of the extracellular matrix proteoglycan versican is the IB4-reactive glycoconjugate associated with these nociceptors. We isolated (by subcellular fractionation and IB4 affinity chromatography) a glycoconjugate from porcine spinal cord tissue that migrated in SDS/PAGE as a single distinct protein band at an apparent molecular mass of > 250 kDa. By using MALDI-TOF/TOF MS, we identified this glycoconjugate unambiguously as a V2-like variant of versican. Moreover, we demonstrate that the IB4-reactive glycoconjugate and the versican variant can be co-released from spinal cord membranes by hyaluronidase, and that the IB4-reactive glycoconjugate and the versican variant can be co-precipitated by an anti-versican immunoglobulin and perfectly co-migrate in SDS/PAGE. Our findings shed new light on the role of the extracellular matrix, which is thought to be involved in plastic changes underlying pain-related phenomena such as hyperalgesia and allodynia.