Phytohemagglutinin-induced Lymphocyte Transformation Research Articles

Effects of an immuno-enhanced diet containing antioxidants in esophageal cancer surgery following neoadjuvant therapy

Neoadjuvant therapy-induced immunological deterioration may be a key factor in postoperative morbidity in patients with esophageal cancer. This study aimed to determine the effects of perioperative feeding with an immuno-enhanced diet on immune competence in patients treated with neoadjuvant therapy followed by surgery. Because an immuno-enhanced diet that contained several antioxidants was used, perioperative oxidative stress and the effects of the immuno-enhanced diet on this stress were also investigated. Of 39 patients with esophageal cancer who underwent similar surgical procedures, 26 patients who received chemotherapy or chemoradiation therapy before surgery were randomly divided into two groups: group 1 (n= 14) was given an immuno-enhanced diet for 5 days before surgery, and group 2 (n= 12) received no enteral feeding products before surgery. Group 3 (n= 13) consisted of patients that did not receive neoadjuvant therapy and received no enteral feeding products before surgery. Several markers for coagulation and fibrinolysis were determined and immunological assessments were performed for each patient. To measure reactive oxygen metabolites and the total antioxidant capacity, diacron-reactive oxygen metabolites (d-ROMs) and OXY-adsorbent tests were performed using a free radical elective evaluator. Significant depression in lymphocyte numbers was observed in groups 1 and 2 before and early after surgery as compared to group 3. Numbers of B cells, CD4/CD8 ratio, and phytohemagglutinin-induced lymphocyte transformation tests were also significantly decreased in groups 1 and 2 on postoperative day 1. Fibrin and fibrinogen degradation products were significantly elevated in group 2 compared to group 1. d-ROMs and OXY-adsorbent test values were elevated before surgery and were decreased transiently early after surgery. Compared to groups 2 and 3, d-ROMs values were significantly lower in group 1 patients throughout the postoperative period, while OXY-adsorbent test values were significantly higher in group 2 patients. Oxidative index was significantly suppressed in group 1 compared to group 3. No significant intergroup differences were observed with regard to morbidity after surgery. Although the baseline levels of immunological function might have been different because of less-advanced cancer stages in group 3, neoadjuvant therapy significantly affected several immunological parameters. Preoperative administration of an immuno-enhanced diet did not significantly prevent neoadjuvant therapy-induced immunological deterioration prior to esophageal cancer surgery. Patients with esophageal cancer had elevated levels of oxidant and antioxidant activities before surgery, which were transiently decreased early after surgery. Although the underlying mechanisms for these perioperative changes are unclear, this study showed that an immuno-enhanced diet containing several antioxidants may reduce oxidative stress following esophageal cancer surgery. After these mechanisms are studied further, oxidative stress control may become another tool for perioperative management to reduce morbidity after esophageal cancer surgery.

Synergistic and antagonistic effects of combinations of cyclosporine A and its metabolites on inhibition of phytohemagglutinin-induced lymphocyte transformation in vitro

Cyclosporine A (CsA) and purified CsA metabolites were tested alone and in combination in cell culture to determine their effects on phytohemagglutinin (PHA)-induced lymphocyte proliferation. CsA was significantly more inhibitory than its metabolites at all concentrations tested (0–1000 ng/mL). CsA exerted maximum inhibition (70% decrease in [ methyl- 3H]thymidine incorporation) at concentrations of 300 ng/mL or greater; metabolites M1, M17, and M21 depressed the response 46, 39, and 23%, respectively, at 300 ng/mL. Metabolites M8, M18, M26, M25, M13, and M203–218 were non-inhibitory. When combinations of M17 and CsA were tested for the effects on PHA-induced lymphocyte transformation, a synergistic effect occurred at combinations of low concentrations of M17 and CsA and an antagonistic effect at the higher concentrations. Of the 49 combinations of CsA and M17 tested, 30 were antagonistic, 16 synergistic and 3 undecided (approaching additivism). When 49 combinations of CsA and the non-immunosuppressive metabolite M8 were tested, 29 of the 49 combinations were synergistic, 17 antagonistic, 1 additive and 2 undecided (approaching additivism). Of the 29 synergistic combinations, 14 were strongly synergistic. The importance of the interaction of CsA and metabolites to the immunopharmacology of CsA therapy is discussed.

Human seminal plasma contains a protein that shares physicochemical, immunochemical, and immunosuppressive properties with pregnancy-associated plasma protein-A.

Pregnancy-associated plasma protein-A (PAPP-A) was detected by RIA in human seminal plasma. This was not due to interference with proteases or binding to seminal plasma proteins, since immunoreactivity was not affected by treatment with protease inhibitors, and the elution of [125I]PAPP-A was not altered by preincubation with seminal plasma. The major component of the seminal plasma PAPP-A coeluted with pure PAPP-A or plasma PAPP-A from pregnant and nonpregnant women. In the RIA, serial dilutions of seminal plasma gave parallel displacement curves to pregnancy plasma. Removing PAPP-A-like material from seminal plasma by adsorbtion to heparin-Sepharose reduced the inhibitory effect of seminal plasma on phytohemagglutinin-induced lymphocyte transformation. The source of seminal plasma PAPP-A-like immunoreactive material remains to be elucidated, but it is unlikely to be the testis, since PAPP-A levels in semen of vasectomized men were similar to those in nonvasectomized men.

A Case of Varicella Pneumonia Treated With Transfer Factor

To the Editor.— We read the case report of disseminated cryptococcosis treated with transfer factor written by Gross et al (240:2460, 1978). We experienced similar results with transfer factor therapy for a disseminated infection involving both skin and lungs that also was not amenable to antibiotic therapy. Report of a Case.— An 11-year-old boy was being followed up for chronic neutropenia and recurrent otitis media at Oklahoma Children's Memorial Hospital and Grady Memorial Hospital. Immunologic evaluation of this patient's condition disclosed decreased serum immunoglobulin level, absence of isoagglutinins, and normal phytohemagglutinin-induced lymphocyte transformation, but absence of delayed hypersensitivity to a battery of common antigens and an average leukocyte count of less than 100/cu mm. With no previous history of the disease, the patient had shown development of typical vesicles of chickenpox. For the first four days of his illness, he experienced a successive crop of vesicles in excess of that

Multiple lymphocyte transformation tests by phytohemagglutinin in healthy individuals: transient episodes of decreased lymphocyte blastogenesis.

146 serial determinations of spontaneous and phytohemagglutinin-induced lymphocyte transformation as measured by tritiated thymidine incorporation, were performed in 24 healthy individuals. 9 persons were studied intensively during a mean period of 40 days and the other 15 underwent random investigations during a period up to 10 months. Transient episodes of significant decreased lymphocyte transformation were revealed in 14 persons (58.4%) with regard to the spontaneous blastogenesis and in 11 subjects (45.8%) with regard to PHA-induced reactivity. The occurrence of the decreased values in PHA responsiveness was significantly higher in the intensively studied group compared to the randomly investigated one (p = 0.001). The probability of detecting one low PHA-related response in a normal subject was 11%. In only half of the cultures with low spontaneous blastogenic response did a simultaneous decrease in PHA-induced lymphocyte transformation occur. In addition to the currently used stimulation index, a new index termed the Blastogenic Cumulative Index is proposed. It is defined as the sum of the logarithms of the dpm values of the spontaneous and the PHA induced lymphocyte transformation. The CI is suggested to describe lymphocyte blastogenic function more accurately, and due to its simplicity warrents further study. Other indices for lymphocyte blastogenesis determination are discussed.

Cell-mediated immunity in pregnant women.

Phytohemagglutinin-induced lymphocyte transformation (PHA-T) was depressed in pregnant women, as compared with that in nonpregnant women. Pregnancy serum had a suppressive action on PHA-T which was enhanced with the advance in pregnancy. Hydrocortisone, progesterone, alpha-fetoprotein and trophoblast-specific antigen, were demonstrated as immune suppressive factors. From these results, it was concluded that cell-mediated immunity might be reduced in pregnant women and that this reduction might be one of the causes for the maintenance of pregnancy.

Lack of effect of azathioprine on phytohemagglutinin-induced lymphocyte transformation and established delayed cutaneous hypersensitivity.

The effect of experimental long-term azathioprine administration on pre-existing in vitro and in vivo manifestations of delayed hypersensitivity was examined. Adult ewes were administered 3 mg/kg/day azathioprine for periods of up to 28 days. Neither phytohemagglutinin (PHA)-induced nor tuberculin-induced lymphocyte transformation was affected. Similarly, pre-existing skin test positivity to tuberculin showed no observable change following drug therapy. These results agree with the majority of those involving clinical situations and indicate that immunosuppression with azathiprine cannot be effectively monitored by either the lymphocyte transformation response to PHA or by changes in the in vivo or in vitro manifestations of pre-existing delayed cutaneous hypersensitivity.

Phytohemagglutinin-induced lymphocyte transformation in liver diseases and in asymptomatic HBsAg carriers.

Phytohemagglutin (PHA)-induced lymphocyte transformation was impaired in acute viral hepatitis. It was significantly correlated with grades of liver cell damage as shown by prothrombin time, GOT, or GPT. It was also lower in drug-induced hepatitis and in prolonged hepatitis than in controls. Of asymptomatic HBsAg carriers, only those with minimal hepatic change showed lower values in stimulation index as well as incorporated radioactivity.

Control of ornithine decarboxylase activity by cyclic nucleotides in the phytohemagglutinin induced lymphocyte transformation

Summary N2,O2′-Dibutyryl guanosine 3′:5′-cyclic monophosphate enhanced the ornithine decarboxylase activity which was induced during lymphocyte transformation by phytohemagglutinin, while N6,O2′-dibutyryl adenosine 3′:5′-cyclic monophosphate tended to inhibit it. The increased ornithine decarboxylase activity was not observed when N2,O2′-dibutyryl guanosine 3′:5′-cyclic monophosphate alone was added to the non-stimulated lymphocytes. In contrast to the dibutyryl derivative, guanosine 3′:5′-cyclic monophosphate did not show any effect on enzyme induction following phytohemagglutinin stimulation.

Inhibition of human lymphocyte blast transformation by streptolysin O.

Abstract Streptolysin O in the hemolytically active form failed to stimulate in vitro lymphocyte blast transformation as measured by thymidine uptake, while streptolysin O inactivated by heating or cholesterol did stimulate thymidine uptake. Hemolytically active streptolysin O was a very effective inhibitor of phytohemagglutinin-induced lymphocyte transformation, but only when the cells were treated first with streptolysin O. Lymphocytes from a PPD-sensitive donor could not be transformed by PPD when pretreated with streptolysin O. The levels of streptolysin O necessary to block transformation (0.5 to 2.0 HU per milliliter) had no measurable effect on cell viability as measured by Evans-Blue dye uptake and did not cause lysis of lymphocytes.

Immune Alterations in Hodgkin's Disease

Delayed hypersensitivity was studied in 103 untreated patients with Hodgkin's disease in all four stages, using six skin test antigens. Forty-three had phytohemagglutininstimulated peripheral leukocyte cultures. Four- to five-year follow-up data relate initial skin test reactivity and lymphocyte transformation to course and survival. Only 11.7% failed to react to any of the six skin tests. Anergy incidence increased with stage. Mumps and 2,4-dinitrochlorobenzene (DNCB) most frequently yielded positive re- actions. Reactivity correlated with the absence of systemic symptoms as well as with histologic type, and did not correlate with course and survival when compared by stage. Nor did phytohemagglutinin-induced lymphocyte transformation correlate with survival, frequency of relapse, or remission duration. Initial delayed hypersensitivity and lymphocyte transformation does not influence prognosis and survival in Hodgkin's disease.

Immune alterations in Hodgkin's disease. Effect of delayed hypersensitivity and lymphocyte transformation on course and survival.

Delayed hypersensitivity was studied in 103 untreated patients with Hodgkin's disease in all four stages, using six skin test antigens. Forty-three had phytohemagglutininstimulated peripheral leukocyte cultures. Four- to five-year follow-up data relate initial skin test reactivity and lymphocyte transformation to course and survival. Only 11.7% failed to react to any of the six skin tests. Anergy incidence increased with stage. Mumps and 2,4-dinitrochlorobenzene (DNCB) most frequently yielded positive re- actions. Reactivity correlated with the absence of systemic symptoms as well as with histologic type, and did not correlate with course and survival when compared by stage. Nor did phytohemagglutinin-induced lymphocyte transformation correlate with survival, frequency of relapse, or remission duration. Initial delayed hypersensitivity and lymphocyte transformation does not influence prognosis and survival in Hodgkin's disease.

Experimental production of hypersensitivity pneumonitis with bagasse and thermophilic actinomycete antigen

Albino New Zealand rabbits and Sprague-Dawley rats were repeatedly exposed to either: (1) ultrasonically aerosolized actinomycete-laden bagasse extract or (2) Micropolyspora faeni antigen via the intratracheal, intravenous, or intramuscular route. Response was evaluated in terms of pulmonary morphologic changes, development of serum precipitins, skin reactivity, and peripheral lymphocyte 3H-TdR incorporation. Some “normal” unaerosolized rats housed in cages with bagasse flooring demonstrated antibagasse precipitins by Ouchterlony gel diffusion and/or varying degrees of peribronchial and perivascular lymphoid hyperplasia. Peripheral lymphocytes from aerosolized rats failed to incorporate significant 3H-TdR in the presence of bagasse extract but bagasse was shown to inhibit phytohemagglutinin-induced lymphocyte transformation. Rabbits immunized repeatedly intratracheally with actinomycete antigen in saline solution developed serum precipitins, delayed skin reactivity, actinomycete induced peripheral lymphocyte 3H-TdR incorporation plus intense alveolar and interstitial pneumonitis characterized by prominent alveolar macrophage proliferation. Rabbits immunized via the intramuscular route in Freund's adjuvant demonstrated identical immunologic findings but no extensive pulmonary lesions. Our results indicate that repeated respiratory tract exposure to thermophilic actinomycete antigen can induce pulmonary mononuclear cell infiltrates, serum precipitins, and antigen-induced peripheral lymphocyte DNA synthesis. The histopathologic lesions and delayed skin reactions to M. faeni in our intratracheally immunized rabbits suggest that cell-mediated (type IV) hypersensitivity is present in this experimental animal model.

Phytohemagglutinin-induced Lymphocyte Transformation: The Relationship to Prognosis of Hodgkin's Disease

Abstract Forty-three untreated patients with Hodgkin’s disease were evaluated between 1965 and 1967 with PHA-stimulated peripheral leukocyte cultures. No correlation was found between PHA-induced lymphocyte transformation at the time of diagnosis and the clinical course of the patient’s Hodgkin’s disease. There is also no correlation between lymphocyte transformation and the histologic pattern of nodular sclerosis. Although PHA-induced lymphocyte transformation appears to be a general index of immunologic status, it has not proven of value as a prognostic sign in Hodgkin’s disease.

The potentiation of phytohemagglutinin-induced lymphocyte transformation by cell-cell interaction; a matrix hypothesis

Two highly purified mitogenic proteinsisolated from commercial preparations of phytohemagglutinin (PHAP) differ greatly in thier hemagglutinating potency. High-titer hemagglutinin mitogen (H-PHAP) binds strongly to red blood cells (RBC) and causes mixed agglutination of lymphocytes and RBC, while low-titer hemagglunitinin mitogen (L-PHAP) binds poorly to RBC and lacks significant mixed RBC-lymphocyte-agglutinating properties. The addition of autologous RBC markedly potentiates the lymphocyte mitogenic capacity of H-PHAP, whereas lumphocyte transformation by L-PHAP is not enhanced. Potentiatioon is most marked at low and intermediate points on the dose-response curve and diminishes as the plateau of transformation is approached. Potentiation is evident at RBC: lymphocyte ratios of 1:10 and reaches a maximum at 5–10:1. Red cell stroma are also capable of potentiation but stromal-free hemolysate preparations are without effect. Both L- and H-PHAP show substantial binding to lymphocytes and platelets. X-irradiated lymphocytes, themselves incapable of mitotic response, potentiate both H- and L-PHAP-induced lymphocte transformation when mixed with viable lymphocytes in a 1:1 ration. The mitogenic activities of both mitogens are also potentiated by the presence of autologous platelets in platelet: lymphocyte ratios of 3–10:1. Higher ratios are less stimulatory, and may even cause inhibition that lymphocyte transformation. These results indicate that lymphocyte transformation is enhanced by cell-cell, or membrane-membrane interaction. Their explanation lies in a consideration of the subunit structure of PHAP molecules, and the ability of potentiating particles to bind from the fluid phase, and present to the lymphocyte surface, a matrix of directionally oriented quadrivalent PHAP molecules for simultaneous and, consequently, more irreversible binding, thus allowing the lymphocyte to pass its threshold for stimulation at concentrations of soluble mitogen which might otherwise be insufficient. These phenomena are pertinent to, and analogous with, the synergistic interaction of thymus-derived, and bone marrow-derived lymphocytes in the initiation and perpetuation of the immune response and support the matrix theory of antigen concentration and presentation as the basis for cellular cooperation in immune reactions.

Inhibition of Phytohemagglutinin-Induced Lymphocyte Transformation by α Globulins; Lack of Correlation with Phytohemagglutinin Precipitation by Serum Proteins

Summary Human α globulin will suppress the in vitro transformation of human lymphocytes induced by two highly purified mitogens isolated from crude phytohemagglutinin preparations. Only one of the mitogen preparations (H-PHAP), however, forms nonspecific precipitates with human serum or isolated α globulin preparations. These results suggest that α globulin does not interfere with phytohemagglutinin-induced transformation by virtue of a direct reaction with the reaction with the mitogen.

Suppression of In Vitro Antigen-Induced Lymphocyte Transformation by Carrageenan, a Macrophagetoxic Agent

Abstract Carrageenan, a high molecular weight sulfated polygalactan with known macrophage toxic properties was added to cultures of normal and sensitized lymphocytes from human peripheral blood and guinea pig lymph nodes to determine its effect on phytohemagglutinin, and antigen-induced 3H-TdR lymphocyte incorporation. Carrageenan was not toxic for guinea pig or human lymphocytes per se and induced no lymphocyte transformation in vitro. With high doses in the range of 1000 µg/ml and above, some decrease in 3H-TdR uptake was noted. Phytohemagglutinin-induced human and guinea pig lymphocyte transformation was not affected by carrageenan but a marked dose-dependent suppression of antigen-induced 3H-TdR lymphocyte uptake was noted in both species. This suppressive effect was not due to reagent or antigen dilution. It was observed when carrageenan was added to cell cultures prior to antigen or up to 6 hr later, but not when added after 17 hr. These findings suggest that carrageenan acts at an early stage of macrophage-antigen processing and are consistent with previous reports of inhibition of antigen-induced lymphocyte transformation using anti-macrophage serum. They provide further evidence that phytohemagglutinin-induced lymphocyte transformation is not mediated by macrophages and that macrophage-lymphocyte interaction occurs with cells from the peripheral blood as well as from lymph nodes.

The interaction of human macrophages and lymphocytes in the phytohemagglutinin-stimulated production of interferon

In studies of 13 normal adults to determine the blood cell types responsible for interferon production induced by phytohemagglutinin, the following observations were made. (a) In cultures containing 96-100% pure macrophages derived from blood monocytes, no interferon was detected in either the presence or the absence of phytohemagglutinin for up to 92 hr. (b) In cultures of 99.5-100% pure lymphocytes, low levels of interferon were detected in the presence, but not in the absence, of phytohemagglutinin. (c) An average fivefold increase in interferon titers occurred when pure lymphocytes were combined with the macrophages in culture with phytohemagglutinin. The peak response of interferon occurred at 68 hr after the initiation of the combined cultures. For maximum response, phytohemagglutinin was required for the duration of the culture, and both cell types in association were necessary. Medium from phytohemagglutinin-stimulated macrophages or lymphocytes could not substitute for the corresponding intact cell. However, frozen-thawed macrophages in combination with lymphocytes and phytohemagglutinin produced an intermediate interferon response. An increase in either cell type produced an increased response in the range studied: lymphocytes, 0.45-1.8 x 10(6) per ml; and macrophages, 0.5-2.1 x 10(5) per ml. Syngeneic fibroblasts, HeLa cells, or mouse macrophages could not substitute for the human macrophages in the combined cultures with phytohemagglutinin. (d) Although all cultures producing interferon showed some degree of transformation (thymidine-(3)H incorporation into deoxyribonucleic acid), no direct correlation between the degree of phytohemagglutinin-induced lymphocyte transformation and the interferon titers was observed.The demonstration of macrophage-lymphocyte interaction in the production of interferon is of interest in view of the known interrelationship of these same cell types in antibody synthesis and cellular immunity.

Effect of human chorionic gonadotropin on in vitro lymphocyte transformation

Depression of in vitro phytohemagglutinin-induced lymphocyte transformation was achieved by the addition of human chorionic gonadotropin (HCG) to the culture system in a concentration simulating the blood level attained during the greater part of pregnancy. HCG may contribute to altered immunologic reactivity in pregnancy, particularly in the early stages, by participating in a local hormonal immunosuppression of maternal lymphocytes in the vicinity of the developing trophoblast.

The effect of carrageenan on the establishment of delayed hypersensitivity.

Carrageenan, a high molecular weight, sulfated polygalactose known to be toxic for macrophages <i>in vitro</i> was utilized in <i>in vivo</i> experiments to further study the role of this cell type in the initiation and expression of delayed hypersensitivity. When administered intraperitoneally to sensitized guinea pigs around the time of skin testing, carrageenan suppressed the expression of existing delayed hypersensitivity reactions to BSA. In addition, when administered at the time of immunization, carrageenan suppressed the development of subsequent delayed hypersensitivity. Carrageenan, <i>in vitro</i> did not appear to be cytotoxic for lymphocytes either structurally or functionally. It did not possess mitogenic properties and did not interfere with phytohemagglutinin induced lymphocyte transformation.