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Adrenergic activation is known to occur in sepsis and after major surgery or trauma. An elevated serum concentration of adrenaline is followed by lymphocytosis in peripheral blood even in splenectomized patients. The purpose of the present study was to clarify the redistribution of lymphocytes in the tissues during adrenaline infusion. Lymphocytes were isolated from 24 rabbits, labelled with indium-111-tropolone and reinjected into the rabbits. The next day the rabbits were anaesthetized. Eight rabbits received 3 micrograms of adrenaline i.v. followed by 0.2 micrograms/min, eight received 300 micrograms of adrenaline i.v. followed by 20 micrograms/min, while eight received a saline infusion and served as a control group. The activity of labelled cells was imaged with a gamma camera and computer before, during and after adrenaline infusion. The activity of the spleen decreased to 90% and 94% of initial values during low and high doses of adrenaline. The activity of the bone marrow decreased to 91% and 96%, respectively, while the activity of the heart/lung and the liver increased to 107% and 106% with the high dose of adrenaline. In peripheral blood the lymphocytes increased 10%. It is concluded that lymphocytes are redistributed from spleen and bone marrow to peripheral blood, lungs and liver during adrenaline infusion in this animal model.

A 56-year-old man with refractory B-cell lymphocytic non-Hodgkin's lymphoma was treated in a Phase II study with interleukin-2 (IL-2) (Roussel-Uclaf, Romainville, France). The patient had involvement of multiple lymph nodes and medullary and peripheral blood (3.6 x 10(9) monoclonal CD19-positive [CD19+] B-lymphocytes/l). After a 5-day cycle of IL-2 treatment, an eightfold increase of the monoclonal CD19+ population was observed (27 x 10(9) monoclonal CD19+ cells). The lymphocytosis decreased dramatically during the second cycle (days 15 to 19) of IL-2 treatment, resulting in 6 x 10(9)/l peripheral lymphocytes, with 5.5 x 10(9) B-lymphocytes. As soon as day 20, peripheral B-cells again increased considerably, with 32 x 10(9) CD19+ cells/l at day 27. The CD19+ population remained monoclonal as assessed by kappa/lambda cell-surface phenotyping and kappa gene rearrangement evaluation. Kinetics of the monoclonal B-lymphocyte response to IL-2 paralleled the natural killer/lymphokine-activated killer and T-cell response, with a 4-day latency period, suggesting an indirect enhancing effect of IL-2. Before and during IL-2 treatment, peripheral B-lymphocytes never expressed detectable levels of the p55 IL-2 receptor. However, the p75 IL-2 receptor was expressed significantly in the IL-2-responsive monoclonal B-cell population. Tumor necrosis factor alpha, a known (in vitro) B-cell tumor growth factor, reached high serum levels during IL-2 treatment. Response evaluation at day 45 showed stability of the lymph node involvement and the marrow lymphocyte infiltrate. At day 45, peripheral B-cell lymphocytosis was 7.5 x 10(9)/l. To the knowledge of the authors, this is the first report of an in vivo IL-2-induced reversible increase of peripheral monoclonal B-cell lymphocytosis.

Pertussis toxin (Ptx), an important adjuvant for inducing certain organ-specific autoimmune diseases in mice, exerts multiple effects upon the immune system. In addition to its adjuvant effects, which include enhancement of delayed-type hypersensitivity and increased antibody production. Ptx elicits a marked lymphocytosis with a concomitant decrease in thymic weight. In vitro studies indicate that Ptx acts directly on thymocytes and that both susceptible and resistant populations exist. It is believed that these susceptible cells are released into the circulation and account, in part, for the T cell component of the lymphocytosis. We have used flow cytometry to analyze the CD4, CD8, and Thy-1 phenotypes of thymic and peripheral T cells from Ptx-treated mice. In the thymus, there is a dramatic decrease in the number of CD4+CD8+ (double positive) cells at all doses tested (0.25, 0.50, and 1.0 microgram) by day 4 after Ptx treatment. The double negative and single positive populations remain relatively constant. Analysis of Thy-1 expression reveals a significant reduction in Thy-1hi thymocytes, with little change in the Thy-1lo population. Thus Ptx primarily affects and depletes, in a dose-dependent fashion, thymic T cells with an immature phenotype. These results mimic those of corticosteroids, although neither prior adrenalectomy nor treatment with the antiglucocorticoid RU486 are able to prevent the effects of Ptx. In the periphery of Ptx-treated animals, the relative increase in the number of CD4+ T cells is more than that of CD8+ T cells. Double positive and Thy-1hi cells cannot be detected in appreciable numbers. These results are consistent with the concept that Ptx may drive immature thymocytes through accelerated maturation for release into the periphery as single positive, predominantly CD4+, Thy-1lo cells. Increased numbers of such cells may in part account for the immunopotentiating effects of Ptx, particularly as they relate to the induction of organ-specific autoimmune disease. Treatment with purified Ptx beta-oligomer fails to elicit any of the responses described above, indicating that the holotoxin is required for such activities.

The study embraces 122 workers from two productions, where the leading chemical noxious in the working environment is benzol. The workers are distributed in groups according to the calculated index of exposure in view of looking for dependence dose-effect. Routine indices of peripheral blood are examined as well as the activity of myeloperoxidase and alkaline phosphatase in leucocytes. An organ-orientated screening, most frequently used for assessing the state of the liver was used. In a large group of workers (mainly from those with high index of exposure) are established early changes in leucocytes: neutropenia, toxic granules, strongly inhibited alkaline phosphatase in the granulocytes and tendency to leukopenia. It was confirmed, that the strongly inhibited alkaline phosphatase, find, which is established also in other workers with benzol exposure, is an early sign for myelotoxic effect of benzol. The biochemical study of workers gives no data for deviations in the functional status of the liver. Single changes in the indices are found characterizing lipid metabolism in workers from Ist risk group of both productions.

A 70-year-old man presented with clonal large granular lymphocytosis of T-suppressor/cytotoxic immunophenotype, neutropenia, paraproteinemia, and proneness to infection. The patient became severely leukopenic during 14 days of chemotherapy with low-dose cyclophosphamide, and remained so after discontinuation of the drug. Clinically, he was thought to have prolonged chemotherapy-induced marrow hypoplasia. At death, 16 days after the last dose of chemotherapy, autopsy confirmed bone marrow hypoplasia and revealed that well-differentiated, polymorphous, and (immunophenotypically and genotypically) polyclonal B-lymphocytes predominated in normal hematopoietic and lymphoid organs. A similar lymphoid infiltrate was intimately associated with multiple ulcers and smooth muscle necrosis in the stomach. These terminal findings resemble B-lymphoproliferative conditions described in certain forms of immune deficiency.

In previous clinical trials, recombinant interleukin-2 (rIL-2) has been infused at high doses over short periods of time to generate lymphokine-activated killer (LAK) cells in vivo. These trials have been limited by severe toxicities, and the immunologic effects of rIL-2 have been transient. The present study was designed to assess the toxicity and immunologic effects of prolonged administration of low doses of rIL-2. In this phase I study, patients with advanced cancer were scheduled to receive intravenous (IV) infusion of rIL-2 without interruption for 3 months in an outpatient setting. Twenty-one patients received rIL-2 at doses ranging from 0.5 x 10(5) to 6.0 x 10(5) U/m2/d. Treatment was extremely well tolerated, and no patient experienced grade 3 or grade 4 toxicity. The lowest dose level (0.5 x 10(5) U/m2/d) did not have demonstrable immunologic activity. At doses of 1.5 x 10(5) and 4.5 x 10(5) U/m2/d, rIL-2 infusion resulted in the specific expansion of natural-killer (NK) cells (sixfold and ninefold increases, respectively, at these two dose levels) without any changes in B cells, T cells, neutrophils, or monocytes. Grade 2 toxicity was observed at the dose of 6.0 x 10(5) U/m2/d, as three patients required interruption of therapy and two patients who completed therapy developed transient hypothyroidism. In patients with increased NK cells, enhancement of non-major histocompatibility complex (MHC)-restricted cytotoxicity and increased generation of LAK cells in vitro were also demonstrated. Therapy with low-dose rIL-2 can be given safely in an uninterrupted fashion for prolonged periods of time in an outpatient setting. This results in selective expansion of NK cells in vivo with minimal toxicity. Further investigation of this schedule for immunomodulation in vivo should be pursued in phase II studies of both malignant and immunodeficient disease states.

Pertussis toxin is known to elicit lymphocytosis in whooping cough patients and experimental animals, by blocking the extravasation of lymphocytes and stimulating their release from lymphoid organs such as the thymus. The mechanisms responsible for these unique effects of PT are not fully understood. The effect of pertussis toxin (PT) on the invasive behavior of human CCRF-CEM T lymphoma cells has been investigated with the use of a monolayer invasion assay (MIA). We had previously found that invasion of murine T lymphoma cells in this model system was correlated with their ability to extravasate and form metastases after i.v. injection in syngeneic animals. We now show that human CEM cells can also penetrate through a precultured confluent monolayer of murine 10T1/2 fibroblast-like cells within a few hours. In a quantitative MIA run over 24 h, PT at concentrations above 10(-14) M inhibited invasion of the CEM cells. In addition, PT stimulated the release ('evasion') of CEM cells that had invaded under the monolayer before the toxin was added. The A subunit of PT was totally inactive, the B subunit had a small residual effect, and reconstitution of the AB complex partially restored the activity. The invasion-inhibiting activity of two different holotoxin preparations and of the subunits perfectly matched their activity in the Chinese hamster ovary cell clustering assay, which is known to depend on a functional AB complex. We suggest that inhibition of monolayer invasion by PT can be used as an in vitro model system to investigate the cellular and molecular mechanisms underlying the lymphocytosis-promoting action of the toxin. Furthermore, the method is sufficiently sensitive to be used for titration of toxin activity. Our data indicate that the ADP-ribosylating activity of the A subunit is indeed required, and that the promotion of lymphocytosis is not elicited by the binding of the B subunit alone.

A variety of side effects have been reported with the use of interleukin-2 alone or in combination with lymphokine-activated killer cells in patients with disseminated neoplasms. The present study was undertaken to determine the effects of high-dose interleukin-2 administration in normal rats. Sprague-Dawley rats were treated with intravenous recombinant interleukin-2 (900,000 IU/kg/day) for 9 consecutive days. Animals were placed in individual metabolic cages, and arterial blood pressure, food intake, body weight, and urine output were monitored. On day 10, animals were killed by exsanguination, various tissues were harvested, and a variety of hematologic and chemical assays were performed. The results were compared with those of placebo-injected normal control and pair-fed groups. The interleukin-2-treated group exhibited anorexia, weight loss, hypotension, anemia, leukocytosis, lymphocytosis, eosinophilia, hypercalcemia, azotemia, and a marked urinary concentration defect. Histologic examination of various tissues revealed widespread infiltration with mono-nuclear cells and eosinophils in most organs, especially in the lungs and liver of interleukin-2-treated animals. Other abnormalities included severe panlobular hepatitis, hepatocellular necrosis, and thymic involution. Renal involvement was mild and consisted of focal interstitial infiltration by mononuclear cells. According to these observations, administration of high-dose interleukin-2 in normal rats results in a score of significant functional, biochemical, and histologic abnormalities.

Following renal transplantation, a patient developed life-threatening granulocytopenia secondary to a specific combination of drugs which are commonly utilized in this setting. Coincident with the depression of granulocytes, an expansion of natural killer cells was seen, which may have been a consequence of immunosuppressive therapy required for allograft retention. Infection with cytomegalovirus may have contributed to both phenomena.

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein composed of an A-protomer and a B-oligomer. There seem to be at least two molecular mechanisms by which IAP exerts its various effects in vivo and in vitro. On the one hand, some of the effects were not significantly affected by acetamidination of the epsilon-amino groups of the lysine residues in the molecule. These include the activities in vitro (1) catalyzing ADP-ribosylation of one of the membrane proteins directly, (2) enhancing membrane adenylate cyclase activity in C6 cells, (3) reversing receptor-mediated inhibition of insulin or glycerol release from pancreatic islets or adipocytes, respectively, and the activities in vivo (4) inhibiting epinephrine-induced hyperglycemia, (5) potentiating glucose-induced hyperinsulinemia, (6) reducing hypertension and increasing the heart rate in genetically hypertensive rats. These activities are concluded to develop as a result of ADP-ribosylation catalyzed by the A-protomer which is rendered accessible to its intramembrane substrate thanks to the associated B-oligomer moiety. Thus, neither the enzymic activity of the A-protomer nor the transporting activity of the B-oligomer needs free amino groups of the lysine residues in the IAP molecule. On the other hand, additional effects of IAP, such as (1) mitogenic, (2) lymphocytosis-promoting, (3) histamine-sensitizing, (4) adjuvant and (5) vascular permeability increasing, were markedly suppressed by acetamidination of the intrapeptide lysine residues. The free epsilon-amino group of lysine would play an indispensable role in the firm (or divalent) attachment of the B-oligomer of IAP to the cell surface that is responsible for development of these activities.

Inoculation of rats i.v. with dextran sulfate or polymethacrylic acid causes lymphocytosis. Electron microscopy of sections of cervical lymph nodes in control rats showed active passage of small lymphocytes through the high endothelial cells (HEC) of the post-capillary venules. In treated rats there was no indication of such passage: the HEC were free of small lymphocytes; and in about 25% of the venules apparently stationary lymphocytes, lacking microvilli, were found appressed to the luminal walls of the HEC.

Chemical modification of amino groups in the molecule of islet-activating protein (IAP), pertussis toxin, resulted in differential modification of biological activities of the toxin estimated in vivo with rats. Acetamidination of epsilon-amino groups of 50% (or more) of lysine residues in the IAP molecule totally abolished the lymphocytosis-promoting activity, but exerted no effects on the epinephrine-hyperglycemia inhibitory activity, of the toxin. Both activities were abolished by acylation of 50% or more of the amino groups probably due to the destruction of the toxin's quarternary structure. In contrast, the subunit assembly of IAP was maintained after exhaustive acetamidination of its lysine residues. The ADP-ribosyltransferase (or NAD-glycohydrolase) activity of the A-promoter (the biggest subunit) of IAP, which is responsible for the principal action of the toxin, enhancing insulin secretory responses and thereby inhibiting epinephrine hyperglycemia, was not affected by acetamindination of lysine residues. Thus, the A-protomer moiety of IAP is not directly involved in, but the amino groups of lysine residues in other subunits are selectively essential for, the development of the toxin-induced lymphocytosis.

The effect of levamisole (LMS) on PPD-induced lymphocyte proliferation in vitro has been investigated in 12 patients with pulmonary tuberculosis and 7 healthy tuberculin-positive control subjects. The augmentative effect of LMS was variable when used directly in vitro and also after pre-incubation of the donor cells with LMS. After ingestion of a single 150 mg dose of LMS, lymphocytes from the control subjects showed significant increased responses in PHA- and PPD-induced lymphocyte proliferation.

Styrene monomer is a greatly used chemical, chiefly in the production of polystyrene. A cross-sectional health survey of 493 production workers was undertaken at the oldest and largest monomer production, polymerization, and extrusion facility in the United States. Relative exposure durations and levels were obtained from occupational histories and corroborated by spot air sampling, blood and fat styrene concentrations and levels of urinary mandelic and phenylglyoxylic acids. Statistically significant differences between the prevalence of abnormalities in high and low exposure groups were found for the following: history of acute prenarcotic symptoms, history of acute lower respiratory symptoms, peroneal nerve conduction velocities, relative lymphocytosis, and elevated gamma glutamyl transpetsidase. The following showed no distinct pattern in prevalence when analyzed by exposure group: chest radiographic changes; indices or restrictive, obstructive and small airway dysfunction; other hepatic and hematological parameters; carcinoembryonic antigen level; sputum cytopathology; radial nerve conduction velocities; and ophthalmological findings. Clinically significant abnormalities were rare.

Islet-activating protein (IAP) pertussis toxin is composed of an A-protomer and a B-oligomer. The B-oligomer binds to the mammalian cell surface via the constituent two dimers (D1 and D2). IAP displays several biological effects including lymphocytosis-promoting, histamine-sensitizing, adjuvant, vascular permeability increasing and mitogenic activities. These activities were markedly suppressed by acetamidination or reductive methylation of the lysine residues in the D2 molecule. The free amino groups of the lysine residues thus play an essential role in the binding of D2 to cells which is responsible for development of these biological activities. In sharp contrast, the islet-activating activity was not prevented by the same chemical modification of the IAP molecule. This activity results from ADP-ribosylation catalyzed by the A-protomer which is rendered accessible to the intramembrane substrate protein thanks to the B-oligomer binding to the cell surface. Free amino groups of the lysine residues played no role in this type of binding of the B-oligomer via D1. Thus, at least two mechanisms underly the diverse biological activities of pertussis toxin.

Analysis of the lipopolysaccharide (LPS, endotoxin) in cell sonicates of four Danish vaccine strains of Bordetella pertussis (3803, 3825, 3843 and 3860) and of purified strain 3803 LPS in sodium dodecyl sulphate-polyacrylamide gel electrophoresis by silver staining, showed identical profiles. The LPS profile revealed a dominant, brownish LPS II band and a minor, faster-migrating, black-stained LPS I band. However, the ratio of LPS I to LPS II in the preparation of purified LPS differed slightly from the cell sonicates. Using marker LPS, the molecular weights of LPS I and LPS II were estimated at 5.4 and 6.0 kD, respectively. Seven different lots of whole cell pertussis vaccine were assayed for LPS in the Limulus Amoebocyte Lysate test and were found to contain 0.9-2.8 micrograms LPS/ml. No significant difference in the content of LPS in similar dilutions of the individual strains was observed. In addition, the distribution of free and cell-bound LPS in four pertussis vaccines was investigated. Most of the LPS was found to exist as free LPS. During several months, the course of both LPS and pertussis toxin (Pt) release in freshly killed B. pertussis preparations was followed. In the first few weeks, 35-50% of the LPS was released and after 5-6 months of storage 60-80% had been released. In contrast, less than 10% of the biologically active pertussis toxin was released during the experimental period. The possibility of producing a safer whole cell pertussis vaccine by reducing the amount of free LPS without reducing the protective value correspondingly is discussed.

Hematological parameters were studied in normotensive and spontaneous hypertensive rats along with those treated with chronic subcutaneous injections of CCl4. The normotensive animals treated with CCl4 demonstrated an erythrocytosis with an increase in the hematocrit levels. A lymphocytosis was seen with neutropenia in both CCl4 treated and untreated normotensive groups. The SHR animals compared to their normotensive counterparts had a lesser degree of lymphocytosis with an increase in the number of neutrophils. There was no blood pressure change in the normotensive CCl4 treated group, however a significant blood pressure difference was observed in the SHR group after CCl4 treatment.

We describe a case of severe sulfasalazine allergy. Exacerbation of the symptoms occurred after unintentional rechallenge with co-trimoxazole, indicating that the reaction was triggered by the sulphonamide component. The clinical picture consisted of generalised adenopathy, hepatitis, high fever and a maculopapular skin rash. A bone marrow biopsy and skin biopsy both showed noncaseating granulomas. The white blood cell count rose to 90.10(9)/l with 40% atypical lymphocytes (plasmacytoid). They were identified by flow cytometry as activated T lymphocytes.

Seventeen patients with terminal malignancy have been entered into a sequential investigation of two doses of continuous infusion recombinant interleukin-2 (bioleukin) given in the setting of a general ward. After an initial experience of a dose of 300 micrograms m-2 in eight patients the remainder received 400 micrograms m-2. Temporary interruption of treatment at the first sign of any serious toxicity led to rapid resolution of side-effects. No patient needed intensive care support, although nine of 17 required temporary interruption of infusion, lasting on average 4 h. Median lymphocyte rebound on day 14 was 3.6 times the pre-treatment level. It remained above pre-treatment levels in four of five patients who had no shown disease progression at day 56 after more than 28 days off treatment. Minor responses occurred in five patients, lasting on average 4 months.

A 38-year-old man presented with a generalized pruritic maculopapular rash, fever, myalgias, and edema of the face and neck. Laboratory examination revealed eosinophilia, atypical lymphocytosis, and abnormal liver function results. The clinical course was characterized by rapid resolution after initiation of steroid therapy and withdrawal of sulfasalazine, which had been started three weeks earlier for abdominal cramping and diarrhea. This clinical picture suggests a drug-induced hypersensitivity reaction to a drug commonly used for inflammatory bowel disease, sulfasalazine.

The fractional production rate of CLL lymphocytes, certainly in the peripheral blood and possibly in the lymph nodes, is often lower than normal. On the other hand, the absolute production in CLL blood and in lymph nodes seems to be considerably increased in most cases. Arguments for an increased lymphocyte life span in CLL, although fragmentary and dispersed, exist. The blood CLL lymphocytes, as normal lymphocytes, exchange rapidly with an extravascular pool of lymphocytes. However, recirculation of lymphocytes--from blood to blood via the lymph node and the thoracic duct--does not proceed normally in CLL. Whether this is related to the B nature of the CLL lymphocyte, to the leukaemic nature of the CLL lymphocyte or to mechanical factors--crowding of the extravascular pool--is still debatable.

The effect of exogenous sex hormones on the cell mediated response in male and female mice has been studied by measuring the subcutaneous growth of HeLa tumour nodules and the variation in the total lymphocyte count. It was found that oestrogen treated male and female mice experienced a profound lymphopenia which was vary rapid in onset. Concurrent with the lymphopenia there was prolongation of HeLa tumour nodule growth in female mice, but not in males. A lymphopenia occurred in androgen treated male mice with subsequent prolongation of HeLa tumour nodule growth, and a lymphocytosis in female mice, with reduction of HeLa tumour nodule growth.

The effects of single and multiple injections of N-acetyl muramyl dipeptide (MDP) on peripheral leukocytes, colony-forming cells (i.e., bone marrow granulocyte-macrophage progenitor cells), and the humoral immune response (to bovine serum albumin) were investigated in mice. Whereas low doses of MDP (0.1 to 1 mg/kg) provoked lymphocytosis, larger doses (10 mg/kg upward) resulted in lymphocytopenia and an increase in the number of young stab neutrophils and monocytes. MDP induced a dose-dependent increase in the number of bone-marrow macrophage progenitor cells, the maximum being reached by a dose around 10 mg/kg. A 50% increase in the maximum effect was produced by a dose around 0.1 mg/kg. The higher the dose, the longer the increase in these progenitor cells persisted. MDP mediated a dose-dependent antibody response to small amounts of bovine serum albumin, correlating with the proliferation of progenitor cells.

A single subcutaneous injection of 0.2 mg epinephrine into healthy human subjects caused a transient lymphocytosis in peripheral blood. Mononuclear cells (MNC), isolated at various times after epinephrine administration, were cultured in the presence of mitogens. The blastogenic responses to pokeweed mitogen (PWM) and phytohemagglutinin (PHA) were significantly reduced for up to 60 min post-epinephrine (p less than 0.05); the response to concanavalin A (Con A) was reduced in the 15-min samples only. All responses returned to pre-injection levels by 120 min post-injection. Removal of adherent monocytes from MNC isolates before culture did not restore normal mitogen responsiveness. When MNC were cultured in the absence of mitogens, there was no difference in survival between pre- and post-epinephrine samples. Incubation of untreated MNC for 2 hr or 18 hr in vitro with various concentrations of epinephrine (10(-5) to 10(-1) mg/ml) had no effect upon the subsequent blastogenic response to mitogens. Other workers have reported that epinephrine administration causes alterations in the composition of the circulating lymphocyte pool. Taken together, these data suggest that the reduction in mitogen responsiveness after epinephrine is the result of changes in the distribution of lymphocyte subclasses in peripheral blood.

The effect of adding 500 micrograms of (2,6-0-dimethyl) beta-cyclodextrin (Me-beta-CD) per ml of Stainer-Scholte (SS) medium in two-day shaker flask cultures of Bordetella pertussis on the production of lipopolysaccharide (LPS) was investigated. The amount of LPS per 10(9) cells found in the supernatants of these cultures was either somewhat reduced or unaffected by comparison with the amounts in cultures grown in SS-medium alone. In addition, the time course of LPS release from cultures of B. pertussis strain 3843 cells during a 96-h growth period in normal and Me-beta-CD-enriched SS medium is described. By using the enriched medium bacterial growth, the production of filamentous haemagglutinin (FHA) and of pertussis toxin (Pt) and the levels of haemagglutination and lymphocytosis-promoting activity were enhanced to various degrees. Measurements made on sedimented whole and on sonicated B. pertussis cells grown in the two media showed no differences in LPS content. The reasons for the reduced/unaffected LPS production are discussed. It has been suggested that an interaction between hydrophobic cavities of the Me-beta-CD molecules and the 'lipid A' part of LPS reduces the reactivity of LPS in the Limulus Amoebocyte Lysate (LAL) assay. This possibility, however, was rejected as the reactivity of Me-beta-CD-spiked purified B. pertussis strain 3803 LPS, compared with unspiked samples, remained unchanged.

Serum antibody responses to the filamentous hemagglutinin and the lymphocytosis-promoting toxin of Bordetella pertussis after vaccination with diphtheria and tetanus toxoids and pertussis vaccine, adsorbed, were assayed by using the enzyme-linked immunosorbent assay. The effect of early immunization, during the first week of life, on the antibody response also was determined. After vaccination, immunoglobulin G (IgG) and IgM directed against both the filamentous hemagglutinin and the lymphocytosis-promoting toxin were detected. Generally, antibody titers increased with subsequent injections and the age of the children. Maternal antibodies against filamentous hemagglutinin and lymphocytosis-promoting toxin were detected in cord blood. The ability of an infant to produce serum IgG anti-lymphocytosis-promoting toxin after vaccination with pertussis vaccine was inversely related to the cord blood serum IgG anti-lymphocytosis-promoting toxin titer at birth. A good antibody response was observed in infants with low cord blood titers, and a poor antibody response was seen in infants with high cord blood values. The IgM anti-lymphocytosis-promoting toxin response was good in groups with both low and high cord blood titer, with no significant difference observed between the two groups. No IgA anti-lymphocytosis-promoting toxin or IgA anti-filamentous hemagglutinin titers were observed in vaccines. IgA antibodies were observed in convalescent sera from two adults and may be presumptive evidence of infection with B. pertussis.

The purpose of this study was to investigate the effect of dose and duration of infusion of recombinant interleukin-2 (IL-2) on toxicity and immunomodulation. In a phase I/II study, IL-2 was administered intravenously (IV) daily for five consecutive days every other week for 4 weeks of treatment to 23 patients with progressive melanoma, renal, colon, or ovarian cancer by one of four regimens: groups I and II received 3 X 10(5) U/m2/d by two-hour or 24-hour infusion, respectively; groups III and IV received 3 X 10(6) U/m2/d by two-hour or 24-hour infusion, respectively. In a subsequent study, six patients (group V) received a single priming cycle of daily IL-2 for five days at 3 X 10(6) U/m2/d in divided 15-minute infusions every eight hours, before undergoing leukapheresis for lymphokine-activated killer (LAK) cell generation. Toxicity was mild with 3 X 10(5) U/m2/d, but severe chills and fever, moderate hypotension (not requiring IV pressors), and weight gain were observed with 3 X 10(6) U/m2/d. Toxicity was also related to the duration of infusion. In group IV (continuous infusion), fluid retention, weight gain, and azotemia were more frequent and severe than in groups III or V, in which the same total dose was administered by two-hour infusion or in three divided 15-minute infusions. IL-2 induced rebound lymphocytosis, which was directly dose-related and significantly higher in group IV (continuous infusion) than in groups III or V. Dramatic increases in the percentage and absolute number of cells expressing the IL-2 receptor were also most pronounced in group IV. With the higher dose of IL-2, LAK cells appeared in the circulation, and natural killer (NK) cytotoxicity was augmented. The results showed that the toxicity and immunomodulation by IL-2 are dose-dependent and are maximal by continuous infusion compared with two-hour or divided every eight hours infusions.

The amounts of endotoxin, histamine-sensitizing factor and lymphocytosis-promoting factor contained in combined diphtheria-pertussis-tetanus and combined diphteria-pertussis vaccines recently produced in Japan were determined and the relationships among them were statistically analyzed. The estimated values in histamine-sensitizing units (HSU) and lymphocytosispromoting units (LPU) per human dose ranged from 0.3 to 6 and from 0.4 to 5, respectively. A fairly wide range of endotoxin contents, which were expressed in body weight-decreasing units (BWDU), was also demonstrated. All the ranges were larger than those expected from experimental error. There was a positive correlation between the HSU and LPU values although the individual HSU-LPU ratios ranged from 0.3 to 3, but the range was markedly larger than the width of the 95% confidence interval of the mean ratio. Negative correlation between BWDU and LPU was also significant, probably because of the relatively large number degrees of freedom, though the coefficient was fairly small; while that between BWDU and HSU was not significant. The frequency distribution of individual values of HSU was divided into two lognormal distributions. With one of the two groups obtained by this subdivision, a distinct positive correlation bewteen HSU and LPU was demonstrated, but with the other group there was little correlation. Some differences between the two groups were also found in the correlations between BWDU and the other two variates. No factors responsible for the separation of the groups have yet been found.

We investigated the effects of carrageenans (CAR) on mouse hematopoiesis, one of the many biologic systems affected by these galactan polysaccharides. Mice were injected intravenously with potassium CAR (K+-CAR) or iota CAR (I-CAR) and studied for 7 or 14 days, respectively, thereafter. Treatment with either compound induces anemia, granulocytosis, and early profound thrombocytopenia. Treatment with I-CAR results in an early lymphocytosis, and both compounds induce lymphopenia by 18 h after treatment. Treatment with either CAR compound is associated with an early moderate reduction in the number of nucleated cells and granulocyte/macrophage colony forming cells (CFUGM) per femur. Both compounds induce splenomegaly, and I-CAR treated mice develop hypoplasia of the thymus by 18 h after treatment. The splenomegaly is associated with intense splenic hematopoiesis and an increase in the number of spleen histiocytes; many of the latter are engorged with metachromatically staining material, most likely CAR. There is a sustained increase in the numbers of spleen CFUGM after treatment with either compound; in the case of I-CAR this may be due to proliferation of CFUGM in this organ, perhaps effected by the increased levels of plasma colony stimulating activity. Although it has been suggested that I-CAR is relatively nontoxic, and, therefore, potentially useful for in vivo studies, our observations indicate that it has profound effects on hematopoiesis which must be considered when planning and interpreting in vivo studies using this compound.

Four-week-old chickens were fed on diets containing 0, 5, 10, 20 and 40 mg corticosterone per kg for eight days. Packed cell volume (PCV), the numbers of granulocytes (G) and lymphocytes (L), the G:L ratio and plasma corticosterone concentration were measured. Plasma corticosterone concentration was increased by dietary corticosterone from day 2 onwards. The number of lymphocytes was increased by serial bleeding but dietary corticosterone depressed the response and caused a dose-related lymphocytopenia from day 1. There was a dose related increase in granulocytes from day 4 and a dose-related increase in G:L ratio from day 1. A dose-related increase in PCV was evident only on days 2 and 4. Correlation coefficients showed little evidence of relationships between plasma corticosterone concentrations and the recorded blood variables.

Pertussigen (Ptx), referred to by many different names, including pertussis toxin, was separated into five polypeptide subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous Tris-glycine buffer system. Under non-reducing conditions, the apparent molecular weights of the polypeptides (mean 10(-3)) were: S1 (26.3), S2 (24.4), S3 (22.7), S4 (12.2), and S5 (11.3). Under reducing conditions, the apparent molecular weights (mean 10(-3)) were: S1 (28.2), S2 (24.8), S3 (24.3), S4 (12.2) and S5 (13.9). The identity of the individual polypeptide subunits was further confirmed by their unique two-dimensional peptide maps. The polypeptides which showed an apparent increase in molecular weight under reducing conditions were those previously found to contain at least two cysteine residues. Reducing conditions also altered the reactivity of S3 and S2 to polyclonal rabbit antibody in electrophoretic transfer (Western) blot analysis. When Ptx was stored in solution at 4 degrees C, S1 and S5 underwent a gradual decrease in apparent molecular weight, as judged by SDS-PAGE. This decrease occurred in three different buffer systems, and was similar to a decrease in apparent molecular weight of S1 and S5 after treatment with the proteolytic enzymes subtilisin or proteinase K. Neither the changes due to storage nor proteolysis affected the activity of Ptx in regard to hemagglutination, lymphocytosis promotion or histamine sensitization. These changes did, however appear to modify the reactivity of S5 in the Western blot. Both the "endogenous" and enzyme-induced changes in S1 and S5 could be stopped by phenylmethanesulfonyl fluoride. These data suggest that S1 and S5 have exposed determinants in the intact Ptx molecule which are readily cleaved by proteases, but have little bearing on the biological activity of the intact molecule. Resistance to inactivation by proteolytic cleavage may help explain the long duration of Ptx activity within in vivo biological systems.

The effect of administration of 0-2 mg of epinephrine (Parke-Davis) on circulatory lymphocytes was investigated in fifteen normal subjects. Blood samples were taken prior to, 10 and 20 min after, subcutaneous injections. Thymus-derived (T) cells were assayed by sheep red blood cell rosettes, bone marrow-derived (B) cells by their complement receptors and immunofluorescent detection of Fc receptors plus surface immunoglobulins. Their percentages prior to injections were 72-2 +/- 1-4, 13-8 +/- 1-1, and 20-3 +/- 1-3. Ten minutes after injections, the absolute concentrations of these cells increased to 164 +/- 14%, 326 +/- 57%, and 272 +/- 45% respectively of the values prior to injection (averages +/- standard errors). Further, when cells with receptors for both sheep red blood cells and complement were assayed by simultaneous rosette technique, they increased from 2-5 +/- 0-4% to 10-5 +/- 1-3% of the lymphocytes. Such changes were also observed in three subjects who had undergone splenectomy more than 2 years previously, but not in four subjects receiving injections of saline instead of epinephrine.

Polmethacrylic acid (PMAA) induces up to a three-fold increase in the lymphocyte population of peripheral blood in rats, goats and calves after intravenous administration. Other routes of administration are less effective. A maximum lymphocytosis is achieved after 3 hr with all doses in excess of 30 mg PMAA/kg body weight; over the next few hours the lymphocyte level declines to normal. Granulocytes increase steadily for the first 7 hr before declining. Multiple doses of PMAA 2 hr apart failed to maintain or significantly alter the lymphocytosis. PMAA was labelled with 125I and 14C, and was traced to various sites in the rat. The greatest accumulation of radioactivity was in the spleen, lungs, liver, kidney, adrenals and mesenteric lymph nodes (with 14C-PMAA). The accumulation appeared more specific for spleen and lymph nodes since there was only a small loss of activity following removal of blood by whole body perfusion. This supports previous findings indicating that these two tissues play a major role in the development of lymphocytosis. Accumulation in the bone marrow may be indicative of stem cell mobilization. The results are discussed in terms of the lymphocytosis-inducing mechanism and the site of action of PMAA and the possible clinical application to ECIB therapy is considered.

Intravenous polymethacrylic acid (PMAA) significantly increases the number of lymphocytes in the blood of the rat. The relationship between dose-effect and lymphocytosis is linear. The lethal dose in 30 days of PMAA is 120 mg/kg b.w. and the half-lethal dose 80 mg/kg b.w. The treatment with 40 mg/kg b.w. intravenous PMAA gives no toxic histological changes either in the lymph organs, the liver or the kidneys. Thus, PMAA appears to be, at present, a most suitable agent by which to provoke experimentally, migration of the reserve lymphocytes into the blood.

More than twenty different polymers, mostly polyanions, were tested in rats for their ability to mobilize lymphocytes into the peripheral blood within 2--3 h. The various polyanions differed in basic structure, in side-chain (size and type), in molecular weight and configuration and in amount and type of anionic charge along the molecule. Neutral polymers and a polycation were also tested for comparison. Some of the polyanions were found to be very effective, others less so and some completely ineffective. Some were also toxic. The basic polymer to which the others were compared was polymethacrylic acid (PMAA), an already recognized mobilizing agent. The best agents were the heparinoids, sulphated polyanions, and the best of these, causing a 3--4-fold increase, were dextran sulphate and polyvinyl sulphuric acid (PVSA). Heparin, although the strongest anticoagulant, was the weakest mobilizer. Some factors that appear capable of modifying mobilization to varying degrees were molecular weight, size and configuration, sulphate content and mode of administration. In the case of PVSA, the smaller molecular weight substance gave a more prolonged lymphocytosis in blood. The high molecular weight substance gave a peak after 2 h, slightly earlier than with PMAA (2--3 h). The administration of protamine chloride to the rat (i.v.) caused an immediate reversal of mobilization, following a course to control values which was essentially identical to the normal decline, only earlier. Dextran sulphate of low molecular weight seems to be the polyanion of choice in subsequent mobilization experiments dealing with determination of the specific mononuclear cell type being mobilized. The single factor that all mobilizing polyanions have in common is a negative molecular charge. It is not yet known exactly how this charge induces the mobilization of mononuclear cells, nor what causes the variability in effectiveness among polyanions.