Mitogenic Lectins Research Articles

Unresponsiveness of CD4−8+/− thymocytes to lectin stimulation in LEC mutant rats

A mutant strain of rat, LEC, shows a novel arrest of T-cell maturation from CD4+8+ to CD4+8-, but not to CD4-8+ cells in the thymus. The responsible mutant locus is designated the thid, which was acted upon in a recessive manner of inheritance. We found that LEC rat thymocytes failed to respond to interleukin (IL)-1, IL-6 and IL-7 in the presence of the mitogenic lectins, Allo A or concanavalin A (Con A). The unresponsiveness appeared to be due to unresponsiveness to the lectin stimulation rather than because of cytokine stimulation. Normal rat CD4-8+/- (consisting of CD4-8+ and CD4-8- thymocytes), CD4+/-8- (consisting of CD4+8- and CD4-8- thymocytes), and CD4-8- thymocyte subsets normally responded to mitogenic stimulation, while CD4+8+ thymocytes did not. In contrast, all LEC rat CD4-8+/-, CD4+/-8-, CD4-8- and CD4-8+ thymocytes did not respond to the mitogenic stimulation, suggesting that the unresponsiveness of the CD4-8+/- thymocytes seems to be responsible for the unresponsiveness of whole thymocytes in LEC rats. LEC rat CD4-8+/- thymocytes normally expressed Con A receptor (R), lymphocyte function-associated antigen-1 (LFA-1), and CD45, which are thought to be important molecules for lectin stimulation. When backcross rats from (F344xLEC)F1xLEC were examined, the phenotype for the thid mutation correlated with the [3H]thymidine deoxyribose (TdR) incorporation level in response to Con A stimulation; thymocytes from backcross rats showing +/thid phenotype responded to Con A stimulation normally, whereas thymocytes from backcross rats showing thid/thid phenotype showed significantly lower responsiveness compared with +/thid rats. However, in WKAH.C-thid congenic rat thymocytes that carry the thid mutation, the responsiveness to mitogenic stimulation was comparable to that of normal rat thymocytes. These results suggest that a defect in responsiveness to mitogenic stimulation in LEC rat thymocytes is controlled by multiple genetic loci and the thid locus is one of the important loci for the development of this abnormal phenotype.

A 70-kilodalton recombinant heat shock protein of Candida albicans is highly immunogenic and enhances systemic murine candidiasis.

The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-gamma), IL-12, and IL-10 but not IL-4 production by CD4+ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-gamma was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-gamma upon in vitro stimulation of CD4+ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.

Immunosuppressive applications of PHA and other plant mitogens.

Phytohemagglutinin was prominent in the evaluation of the immunosuppressive effects of mitogenic lectins that started in 1965 and continued for two decades. Basic studies elucidated the inhibitory actions of PHA on humoral and cellular immune responses in mice, rats, and guinea pigs. Some suppressive effects of this and other mitogens including lentil lectin and Con A were demonstrated on renal, skin, pancreas, and heart allograft rejections in mice, rats, and dogs. In addition to their inherent suppressive activities, these substances have been shown to potently augment the suppression generated by conventional agents. More relevant to tolerance-inducing modulations, the Rigas group showed that in vitro incubation of the parental spleen cells with PHA in the F1 hybrid model before giving them to the F1 mouse virtually abolished the GvH responses. Their explanation was that the donor cells were rendered unresponsive by their reversion to an immature state in which they lost the essential surface receptors. Similar spleen cell suppression was generated by systemic administration of PHA to the parental donor prior to their administration. A method is proposed here for establishing tolerance to either newly introduced or firmly established antigens applying the L4 isolectin of PHA to make non-reactive all T lymphocytes that remain functional in conjunction with full suppression by conventional agents. During the vulnerable period of drug-induced suppression, the host would be protected from infection and bleeding by full nonspecific proliferative activation of the lymphoid and myeloid systems. The establishment of allograft tolerance by preemptive inhibition of responses to newly introduced transplant antigens would be easier to achieve than the reversal of firmly established responses to antigens involved in GvH reactions and autoimmune diseases. The studies of von Boehmer and Kisielow in a transgenic mouse model confirmed that clonal deletion does occur in the thymus whereby corresponding specific thymocytes are destroyed when they encounter self-antigens. Their work suggested that tolerance to self-antigens might be established if immune responses against putative antigen peptides were blocked sufficiently that they would be released to reach the central or peripheral residence sites of their immunologically reactive clones for deletion to occur. This approach to suppressive therapy would be useful for managing the problems of allograft rejection, GvH reactions, and autoimmune disorders even if they fell short of generating complete tolerance. Suppressive treatment with mitogens would also be indicated for the immune-related group of aplastic anemias, but a discussion of these disorders will be deferred to a separate article because of certain aberrations they present.

Granzyme Release and Caspase Activation in Activated Human T-Lymphocytes

Recently it has been reported that caspase-3 activation occurs in stimulated T-lymphocytes without associated apoptosis (Miossec, C., Dutilleul, V., Fassy, F., and Diu-Hercend, A. (1997) J. Biol. Chem. 272, 13459-13462). To explore this phenomenon, human peripheral blood lymphocytes (PBLs) were stimulated with mitogenic lectins or anti-CD3 antibody, and the proteolytic processing of different caspases and caspase substrates was analyzed by immunoblotting. Proteolytic processing of caspases-3 and -7 and the caspase substrates poly(ADP-ribose) polymerase, GDP dissociation inhibitor, and PKCdelta was observed when PBLs were activated in vitro, and lysates were prepared using RIPA buffer which contains 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS. In contrast, when a lysis buffer containing 2% SDS was used, the caspases remained in their zymogen pro-forms, and no proteolytic processing of caspase substrates was detected. Moreover, in experiments using intact cells and a cell-permeable fluorigenic caspase substrate, no caspase activity was observed in activated T-cells, whereas it was clearly detected when PBLs were treated with the apoptosis-inducing anticancer drug etoposide. Since the granzyme B is a direct activator of caspase-3 and its expression is induced following T-cell activation, we tested the effects of anti-GraB, an engineered serpin that specifically inhibits GraB. When the activated T-lymphocytes were lysed in RIPA buffer containing anti-GraB, no proteolytic processing or activation of caspase-3 was observed, strongly suggesting that release of GraB or similar proteases from their storage sites in cytotoxic granules during the lysis procedure is responsible for caspase activation. These findings demonstrate that T-cells do not process caspases upon activation and caution about the method of cell lysis used when studying granzyme-expressing cells.

The Raf-1/Mitogen-Activated Protein Kinase Kinase-1/Extracellular Signal-Regulated-2 Signaling Pathway as Prerequisite for Interleukin-2 Gene Transcription in Lectin-Stimulated Human Primary T Lymphocytes

ABSTRACT. It has been shown that stimulation of lymphoid cells causes the activation of the extracellular signal-regulated-2 (ERK-2) which activates nuclear factor of activated T cells (NF-AT), a transcription factor involved in the regulation of interleukin-2 (IL2) gene transcription. ERK-2 is activated via a kinase cascade initiated by activation of the G protein p21Ras followed by phosphorylation and activation of Raf-1 and mitogen-activated protein kinase kinase-1 (MEK-1). Activation of this pathway has been described primarily in human T cell lines; however, using primary T lymphocytes from transgenic mice, a recent study has shown that a blockade of this cascade did not perturb lymphocyte stimulation and proliferation. In the present paper, we studied in human primary T cells the possible involvement of the Raf-1/MEK-1/ERK-2 pathway upon stimulation by jacalin, a mitogenic lectin which specifically stimulates CD4 + lymphocytes. We show here that the mitogen-activated protein (MAP) kinase pathway was stimulated in human purified lymphocytes upon activation with jacalin. Moreover, activation of this pathway appeared to be essential, since its blockade by a specific inhibitor of the MEK-1 kinase abolished IL2 gene transcription; in contrast, in T cells stimulated with phytohemagglutinin M(PHA), another potent T cell mitogenic lectin, blockade of MEK-1 reduced but did not totally inhibit either ERK-2 phosphorylation or IL2 mRNA expression. This shows, as already suggested, that another pathway in addition to the Raf-1/MEK-1/ERK-2 kinase cascade could be triggered in T cell activation. Jacalin stimulation therefore appeared to be a good model for the specific activation of the MAP kinase pathway in human primary T lymphocytes, which would allow the characterisation of drugs specifically targeted to this particular pathway.

Peanut ingestion increases rectal proliferation in individuals with mucosal expression of peanut lectin receptor

Background & Aims: The Thomsen–Friedenreich blood group antigen (galactose β1,3- N-acetyl galactosamine α-) acts as an oncofetal antigen in the colonic epithelium, with low expression in normal adult epithelia but increasing to fetal levels of expression in hyperplasia or malignancy. Peanut lectin is one of the commonest dietary lectins that binds this antigen. The aim of this study was to determine whether peanut ingestion can alter rectal epithelial proliferation. Methods: Thirty-six patients with normal colonic mucosa consumed 100 g of peanuts each day for 5 days. Rectal mitotic index was measured before and after ingestion, and changes in proliferation were correlated with immunohistochemical detection of lectin receptor expression by colonocytes and fecal lectin activity as measured by hemagglutination assay. Results: Peanut ingestion caused a 41% increase in rectal mucosal proliferation in individuals with macroscopically normal mucosa who express TF antigen in their rectal mucosae (10 of 36 patients studied). The proliferative response correlated with fecal hemagglutinating activity, and peanut lectin could be shown immunohistochemically within the rectal mucosa. Conclusions: The common expression of galactose β1,3- N-acetyl galactosamine α- by hyperplastic and neoplastic epithelia may therefore be functionally important because it allows interaction with mitogenic dietary lectins. This could be an important mechanism for the association between diet and colorectal cancer. GASTROENTEROLOGY 1998;114:44-49

Tumor Necrosis Factor-alpha and Interferon-r Secretory Capacity of Mononuclear Leukocytes after Incubation in Patient with Acute Myocardial Infarction

Background:Studies of human coronary plaque specimens have shown that T lymphocytes and macrophages are present in all types of lesions, from fatty streaks to advanced plaques. There is growing evidence for a pathogenic role for immune response in progression of atherosclerosis. This study was designed to investigate cytokine production by mononuclear leukocytes from patients with myocardial infarction. Method:We measured the kinetics of secretion of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) by mononuclear leukocytes from 8 control subjects and 12 patients with acute myocardial infarction. Mononuclear leukocytes were isolated and incubated with plant lectin mitogen concanavalin-A for 24 and 48 hours. TNF-α and IFN-γ secretions were measured by ELISA. Results:There were no significant differences between TNF-α and IFN-γ secretions by mononuclear leukocytes at and before 24 hours of incubation from both patients and control subjects, but TNF-α and IFN-γ secretions at 48 hours of incubation were higher (p<0.005, p<0.05) in patients when compared with control subjects. TNF-α and IFN-γ secretions by mononuclear leukocytes after incubation correlated with the peak level of creatine phosphokinase (CK) and CK-MB. Conclusion:Increased cytokine secretory capacity of mononuclear leukocytes may be due to the acute inflammatory response of myocardial infarction. Further trials may be needed to determined the effects of increase in secretory capacity of mononuclear leukocytes before myocardial infarction. (Korean Circulation J 1998;28(4):586-591)

Agonist-induced morphologic decrease in cellular D1A dopamine receptor staining.

The distribution of D1A dopamine (DA) receptor proteins was assessed by using subtype specific antireceptor antisera after acute DA exposure. The immunofluorescent staining of D1A DA receptor protein expression was examined in (1) stably transfected Chinese hamster ovary (CHO) cells, (2) primary striatal cell cultures, and (3) rat striatal brain slices. After agonist exposure as brief as 2 min and as long as 60 min, profound loss of immunofluorescent D1A receptor protein staining occurred in each paradigm. Additionally in the tissue slice, immunofluorescent neuropil staining for the receptor protein also was attenuated. The DA-induced alteration in receptor protein staining was blocked by the antagonist (+)-butaclamol and by the selective D1-family antagonist SCH 23390. Receptor staining patterns reverted back to the control immunofluorescent distribution within 15 min after removing the agonist from the bath. Immunofluorescence for the second-messenger cyclic AMP increased at all DA exposure times in the three experimental paradigms, was blocked by D1-family antagonists, and decreased to basal staining after brief recovery periods. This demonstrated the functional integrity of the D1A receptor in target cells. Pretreatment with the mitogenic plant lectin concanavalin A blocked the immunofluorescent decrease in receptor staining but not the elevation of the second messenger, indicating a morphologic distinction in these two events, parallel to other biochemical reports. The data suggested that a morphologic basis of acute homologous D1A DA receptor desensitization may be transposition of membrane-surface receptors to a transiently unavailable, intracellular compartment. This finding is supported by specific fluorescence incorporation of FM1-43, used as a marker of endocytosis, in CHO cells treated with DA.

Agonist‐induced morphologic decrease in cellular d1A dopamine receptor staining

The distribution of D1A dopamine (DA) receptor proteins was assessed by using subtype specific antireceptor antisera after acute DA exposure. The immunofluorescent staining of D1A DA receptor protein expression was examined in (1) stably transfected Chinese hamster ovary (CHO) cells, (2) primary striatal cell cultures, and (3) rat striatal brain slices. After agonist exposure as brief as 2 min and as long as 60 min, profound loss of immunofluorescent D1A receptor protein staining occurred in each paradigm. Additionally in the tissue slice, immunofluorescent neuropil staining for the receptor protein also was attenuated. The DA-induced alteration in receptor protein staining was blocked by the antagonist (+)-butaclamol and by the selective D1-family antagonist SCH 23390. Receptor staining patterns reverted back to the control immunofluorescent distribution within 15 min after removing the agonist from the bath. Immunofluorescence for the second-messenger cyclic AMP increased at all DA exposure times in the three experimental paradigms, was blocked by D1-family antagonists, and decreased to basal staining after brief recovery periods. This demonstrated the functional integrity of the D1A receptor in target cells. Pretreatment with the mitogenic plant lectin concanavalin A blocked the immunofluorescent decrease in receptor staining but not the elevation of the second messenger, indicating a morphologic distinction in these two events, parallel to other biochemical reports. The data suggested that a morphologic basis of acute homologous D1A DA receptor desensitization may be transposition of membrane-surface receptors to a transiently unavailable, intracellular compartment. This finding is supported by specific fluorescence incorporation of FM1-43, used as a marker of endocytosis, in CHO cells treated with DA. Synapse 27:313–321, 1997. © 1997 Wiley-Liss, Inc.

Inhibition of human lymphocyte proliferation by nitric oxide-releasing oxatriazole derivatives

The effects of novel nitric oxide (NO)-releasing oxatriazole derivatives GEA 3162 and GEA 3175 were studied on cell proliferation and cGMP synthesis in human peripheral blood mononuclear cells stimulated with a lectin mitogen concanavalin A. GEA 3162 (1–30 μM) and GEA 3175 (3–30 μM) inhibited mononuclear cell proliferation in a dose-dependent manner being more potent than the earlier known NO-donor S-nitroso- N-acetylpenicillamine. The inhibitory action was more pronounced when submaximally stimulating concentrations of concanavalin A (0.1 and 1 μg/ml) were used and no inhibition was seen when concanavalin A concentrations were increased up to 10 μg/ml. The antiproliferative concentrations of GEA 3162, GEA 3175 and S-nitroso- N-acetylpenicillamine induced a rapid and transient increase in cGMP production in mononuclear cells cultured in the presence of concanavalin A. Both the antiproliferative action and the increased cGMP production were attenuated when red blood cells were added into the cultures indicating that NO is responsible for both of these actions. An analogue of cGMP, 8-bromo-cGMP (0.1–3 mM) reduced concanavalin A-induced proliferation in a dose-dependent manner suggesting that cGMP may be involved in the antiproliferative action of NO-donors. NO-releasing compounds have immunosuppressive actions which offer therapeutic possibilities and should be kept in mind as potential adverse events when these compounds are used in other indications.

MRNAs ENCODING CCKB BUT NOT CCKA RECEPTORS ARE EXPRESSED IN HUMAN T LYMPHOCYTES AND JURKAT LYMPHOBLASTOID CELLS

We previously reported the existence of pharmacologically related gastrin/CCKB type receptors (CCKB-R) in a variant of Jurkat T lymphoblastoid cells (JK(CD3 −CD4 +)). We studied here the expression of mRNAs encoding CCKA and CCKB receptors in various human white cells by means of Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Using CCKB-R specific primers, we detected a significant expression of CCKB-R mRNA in JK(CD3 −CD4 +) cells. These transcripts were also expressed, at a lower level, in two other Jurkat clones (JK(CD3 +CD4 −) and JK(CD3 +CD4 +)), in peripheral blood lymphocytes (PBL) and in purified CD4 + and CD8 + lymphocytes. Activation of Jurkat cells and PBL by T cells mitogenic lectins (jacalin, phytohemaglutinin) did not modify CCKB-R mRNA expression. In all these cells, using CCKA-R specific primers, we could not amplify any specific cDNA fragment corresponding to this receptor. Neither CCKB-R nor CCKA-R mRNAs could be detected in monocytic cells. Our data show for the first time a constitutive expression of CCKB-R transcripts in lymphoid cells. Moreover, the modulation of immunocyte functions by cholecystokinin-related peptides could occur through CCKB-R rather than CCKA-R and affect lymphocytes rather than monocytes.

The Viscum album preparation Isorel inhibits the growth of melanoma B16F10 by influencing the tumour-host relationship.

The aim of this study was to analyse whether Viscum album (mistletoe; Isorel) modulates the tumour-host relationship and whether this might be a basic mechanism of the antitumorous activity of the drug. The effects of a single intraperitoneal injection of the drug (100 mg/kg single 'planta tota' dose) were analysed for mice-bearing melanoma B16F10 growing in the hind limb. Injection of Isorel reduced the size of the tumour and caused abundant tumour necrosis with inflammatory response, oedema and destruction of the malignant tissue. Furthermore, the lymphocytes of saline-treated tumorous mice were not able to respond to the mitogenic lectin concanavalin A in vitro, while those of mistletoe extract-treated mice showed high reactivity too the mitogen, but only if cultured in the medium supplemented with the plasma of the mistletoe extract-treated mice. Moreover, melanoma cells exposed to the mistletoe extract were more sensitive to the cytotoxic activity of the lymphocytes than the control tumour cells, particularly in the presence of the plasma of mistletoe extract-treated mice. The plasma itself, however, did not show any cytotoxic activity. These results indicate that the antitumour activity of the mistletoe drug is due to a modulation of the tumour-host relationship, mediated by direct cytotoxicity of the drug to tumour cells and/or through a potentiation of immune response by certain, as yet unidentified, growth modifying humoral factors of the host.

Immune system dysfunction during exposure to poult enteritis and mortality syndrome agents

Poult Enteritis and Mortality Syndrome (PEMS) is a condition of yet undefined etiology. Affected flocks may exhibit 100% morbidity with mortality up to 50% or more between 2 to 4 wk of age. The current study reports the immune status of poults experimentally infected with PEMS agent(s) in various trials. When compared with the unchallenged controls, PEMS-infected poults had significant atrophy of the bursa (up to 2-fold), thymus (up to 11-fold), and spleen (up to 2-fold) (P < or = 0.05). When challenged with SRBC, PEMS-infected poults had 1 to 2 log2 lower anti-SRBC antibody titers than the controls (P < or = 0.05). Responsiveness to a mitogenic lectin, phytohemagglutinin-P, was reduced significantly in PEMS poults (P < or = 0.05). These data show that the immune system of the poults is compromised significantly during PEMS infection in terms of lymphoid organ integrity and humoral and cell-mediated immunity. These findings imply, therefore, that immune dysfunction may contribute to the mortality observed during PEMS outbreaks.

Fluorescence depolarization as an early measure of T lymphocyte stimulation

We have used the Cellscan, an apparatus capable of measuring optical properties of individual cells, to study changes in fluorescence polarization associated with T cell stimulation. We show that the fluorescence polarization of human peripheral blood lymphocytes (PBL) labeled with fluorescein diacetate (FDA) is markedly reduced upon exposure to the mitogenic lectins phytohemagglutinin (PHA), concanavalin A (ConA), or to phorbol esters. Methyl α- d-mannopyranoside ( αMM) is able to reverse the depolarizing effect induced by ConA as long as the cells are not committed to proliferate. H7 and staurosporin, both inhibitors of protein kinase C (PKC), inhibit the depolarization induced by PHA. The mitogen-induced depolarization is dependent on metabolic energy. The results support the use of fluorescence depolarization of FDA-labeled PBL, monitored by the Cellscan, as a sensitive means of measuring early lymphocyte stimulation.

Abnormalities in CD69 expression, cytosolic pH and Ca2+ during activation of lymphocytes from patients with systemic lupus erythematosus.

Several immuno-regulatory abnormalities have been described in SLE patients. T cell dysfunction in SLE includes defective in vitro proliferative responses to several stimuli, reduced IL-2 production and a poor helper function. It has been widely proposed that this defective T cell immunoregulatory function has a key role in the hyperactivity of B cells and auto-antibody production in SLE. However, it has not been elucidated whether or not this cell dysfunction is intrinsic to lymphocytes or is due to other factors such as anti-lymphocyte auto-antibodies. In this study we have evaluated some important early cell activation events in T and non-T lymphocytes from patients with systemic lupus erythematosus (SLE). Peripheral blood lymphocytes from SLE patients and controls were isolated. The intracellular pH (pHi), cytosolic calcium (Ca2+i) and CD69 expression were determined by spectrofluorometry and flow cytometry. Modifications of these parameters in response to protein kinase C (PKC) activators, mitogenic lectins and calcium ionophores were also studied. We found a significant reduction in the increase of pHi in response to PKC activators (PMA) in SLE cells. In addition, the induction of CD69 expression by PMA was significantly lower in T cells from SLE patients. By contrast, freshly isolated non-stimulated SLE cells exhibited a significantly higher pHi, as well as an increased baseline expression of the early cell activation antigen CD69. On the other hand, the increase in Ca2+i in response to a Ca2+ ionophore (4Br-A23187) or thapsigargin in Ca(2+)-free solutions, was smaller in SLE lymphocytes. We concluded that T cells from SLE patients exhibit abnormalities in several key early cell activation events (pHi, Ca2+i and CD69 expression). These abnormalities could have an important role in the T cell dysfunction observed in SLE. The presence of T cells with a preactivated phenotype in the peripheral blood of SLE patients, could be a reflection of the ongoing autoimmune phenomena that is occurring in these patients.

Theoretical benefits of mitogen applications for HIV-1 infections.

Ideal treatment of HIV-1 infections should include an agent that can reverse the capacity of the virus to evade destruction by hiding in sanctuaries and by frequently mutating the epitopes it displays. The rapid proliferation of virions during the years of symptomatic quiescence obligates rapid replacement of CD4+ lymphocytes that leads to a gradual attrition of the T lymphocytes needed to control infections. In vitro evidences suggest that, given systematically, certain mitogenic lectins would interfere with HIV-1 invasion of CD4+ cells by blocking gp120 molecules on the viral membrane before activating T lymphocytes subsequent to binding with their Ti/CD3 molecules. The nonspecific nature of antiviral effector cells generated by this activation should circumvent HIV-1 mutations at the same time it reconstitutes depleted T lymphocytes, stimulates myelopoiesis, and reinforces resistance to malignancies and infections prevalent with the immunodeficiency state. Properly coordinating these effects with appropriate combinations of reverse transcriptase and protease inhibitors could theoretically expedite complete elimination of HIV in a timely fashion that shorten the required treatment duration and excludes the detrimental effects of virus mutations. The proper sequence of this treatment should be maximum reduction of the HIV-1 load with drug combinations, control of complicating infection by other means to reduce mitogen-induced tissue necrosis, and addition of systemic PHA-L4 administration regulated to maintain a 5-10 micrograms/mL serum concentration. The antiviral regimen should be continued an undetermined time beyond when HIV-1 is no longer detectable, and systemic L4 administration until satisfactory immunologic and hematologic competences are re-established. Partially-matched mitogen-activated adoptive leukocyte therapy might be additionally helpful.

Therapeutic immunostimulating effects of plant mitogens exemplified by the L4 isolectin of PHA.

A sizable body of evidence has accumulated showing that the characteristics of certain plant mitogens should endow them with valuable immunomodulating effects. Immune stimulation would be the fundamental function operating through the broad consequences of nonspecific T cell activation following binding with their CD3 or CD2 molecules. Based on in vitro evidences that PHA, operating through the LDCC pathway, might kill any tumor target if it remains present in adequate concentration, the administration of mitogens for cancer therapy would be rational, and the same mechanism should also justify these agents for treatment of certain infections. Being nonerythroagglutinating, although leukoagglutinating in higher concentrations, PHA-L4 serves as a suitable model for immunostimulating activities of the mitogens that can be applied directly or as in vitro activators of adoptive leukocytes. The PHA skin test can be utilized to gauge the serum levels of inhibitory glycoproteins that become elevated in a number of the disorders treatable by the mitogen, thus facilitating necessary dosage modifications. While PHA is the only mitogenic lectin that has been tested clinically, Con A and PWM are the two most widely studied among the alternatives, with others also available and many undoubtedly awaiting discovery. The development of practical methods for producing industrial quantities of nonagglutinating mitogens in pure form should be the goal, and accomplishing the latter might also answer certain hypersensitivity concerns.

Reactive oxygen species and nitric oxide in viral diseases.

Metabolites derived from superoxide (O2.-) and nitric oxide (NO.) play an important role in antimicrobial and antitumoral defense, but may also harm the host. Low levels of such metabolites can also facilitate viral replication because of their mitogenic effects on cells. Most viruses grow better in proliferating cells, and indeed, many viruses induce in their host cell changes similar to those seen early after treatment with mitogenic lectins. Influenza and paramyxo-viruses activate in phagocytes in the generation of superoxide by a mechanism involving the interaction between the viral surface glycoproteins and the phagocyte's plasma membrane. Interestingly, viruses that activate this host defense mechanism are toxic when injected in the bloodstream of animals. Mice infected with influenza virus undergo oxidative stress. In addition, a wide array of cytokines are formed in the lung, contributing to the systemic effects of influenza. Oxidative stress is seen also in chronic viral infections, such as AIDS and viral hepatitis. Oxidant production in viral hepatitis may contribute to the emergence of hepatocellular carcinoma, a tumor seen in patients after years of chronic inflammation of the liver. Antioxidants and agents that downregulate proinflammatory cytokines and lipid mediators may be a useful complement to specific antiviral drugs in the therapy of viral diseases.

Contribution of stabilizing agents present in intravenous immunoglobulin preparations to modulation of mononuclear cell proliferation in vitro.

Intravenous immunoglobulin (IVIG) preparations have been shown to suppress lymphocyte proliferation in vitro. This study aimed to investigate the effects of an IVIG produced by fractionation/DEAE-Sephadex adsorption on lymphocyte proliferation in vitro, with particular reference to contributions of the stabilizing agents present in the IVIG to the modulation of mononuclear cell proliferation. It was found that glycine significantly inhibited stimulation by the mitogenic lectin phytohaemagglutinin (PHA; 58% inhibition, P < 0.01). Glucose and human albumin also reduced the response to PHA but to a lesser extent (20% and 30%, respectively). In further experiments the effects of sucrose and maltose, two disaccharides used as stabilizing agents in IVIG preparations, were studied. Three doses were used (2.5 mM, 25 mM and 250 mM), representing levels likely to be found in vivo after infusion of IVIG at immunotherapeutic doses, on four different proliferative stimuli. Maltose was found to inhibit proliferation to PHA, anti-CD3 and PMA in a dose responsive manner. Sucrose also inhibited proliferation to these stimuli, but a dose response was not observed. For both sugars, only the highest dose (250 mM) inhibited the proliferative response of mononuclear cells to PMA and a calcium ionophore (ionomycin). The repurified IgG component of the IVIG preparation did not inhibit PBMC responses to PHA in this system. Kinetic analyses, in which the sugars were added 24 h after proliferative stimuli, indicated that both sugars still inhibited responses to PHA, and, to a lesser extent, PMA, but only maltose inhibited the anti-CD3 response. These findings show that stabilizing agents currently found in commercial IVIG preparations make a significant contribution to modulating mononuclear cell proliferation and need to be considered when assessing the immunomodulatory role of IVIG in vitro.

Purified protein from Salmonella typhimurium inhibits the interleukin-2 response of murine splenic T-lymphocytes activated with anti-CD3 antibody.

Previously, we demonstrated that the immunosuppression induced by a purified preparation of Salmonella typhimurium-derived inhibitor of T-cell proliferation (STI) can be observed in terms of suppression of the proliferation of murine spleen cells stimulated with a mitogenic lectin. In the present study, I observed that STI inhibited the interleukin-2 (IL-2) response of purified murine splenic T lymphocytes stimulated with anti-CD3 antibody. The flow cytometric analysis of IL-2 receptor (IL-2R) expression on T cells showed that STI specifically suppressed the expression of IL-2R beta and IL-2R gamma. Furthermore, when the IL-2-dependent T-cell line CTLL-2 was incubated with STI, the growth of CTLL-2 cells was significantly inhibited. These results suggest that the target cells for STI are T cells themselves, and that the suppression of T-cell proliferation induced by STI might involve a defect in the IL-2 receptor (IL-2R) function of T cells.