Increased ß-haemolytic group A streptococcal M6 serotype and streptodornase B-specific cellular immune responses in Swedish narcolepsy cases.

BACKGROUND: Type 1 narcolepsy is a neurological disorder characterized by excessive daytime sleepiness and cataplexy associated with the HLA allele DQB1*06:02. Genetic predisposition along with external triggering factors may drive autoimmune responses, ultimately leading to the selective loss of hypocretin-positive neurons. OBJECTIVE: The aim of this study was to investigate potential aetiological factors in Swedish cases of postvaccination (Pandemrix) narcolepsy defined by interferon-gamma (IFNγ) production from immune cells in response to molecularly defined targets. METHODS: Cellular reactivity defined by IFNγ production was examined in blood from 38 (HLA-DQB1*06:02(+) ) Pandemrix-vaccinated narcolepsy cases and 76 (23 HLA-DQB1*06:02(+) and 53 HLA-DQB1*06:02(-) ) control subjects, matched for age, sex and exposure, using a variety of different antigens: ß-haemolytic group A streptococcal (GAS) antigens (M5, M6 and streptodornase B), influenza (the pandemic A/H1N1/California/7/09 NYMC X-179A and A/H1N1/California/7/09 NYMC X-181 vaccine antigens, previous Flu-A and -B vaccine targets, A/H1N1/Brisbane/59/2007, A/H1N1/Solomon Islands/3/2006, A/H3N2/Uruguay/716/2007, A/H3N2/Wisconsin/67/2005, A/H5N1/Vietnam/1203/2004 and B/Malaysia/2506/2004), noninfluenza viral targets (CMVpp65, EBNA-1 and EBNA-3) and auto-antigens (hypocretin peptide, Tribbles homolog 2 peptide cocktail and extract from rat hypothalamus tissue). RESULTS: IFN-γ production was significantly increased in whole blood from narcolepsy cases in response to streptococcus serotype M6 (P = 0.0065) and streptodornase B protein (P = 0.0050). T-cell recognition of M6 and streptodornase B was confirmed at the single-cell level by intracellular cytokine (IL-2, IFNγ, tumour necrosis factor-alpha and IL-17) production after stimulation with synthetic M6 or streptodornase B peptides. Significantly, higher (P = 0.02) titres of serum antistreptolysin O were observed in narcolepsy cases, compared to vaccinated controls. CONCLUSION: ß-haemolytic GAS may be involved in triggering autoimmune responses in patients who developed narcolepsy symptoms after vaccination with Pandemrix in Sweden, characterized by a Streptococcus pyogenes M-type-specific IFN-γ cellular immune response.

Debridement for surgical wounds.

BACKGROUND: Surgical wounds that become infected are often debrided because clinicians believe that removal of this necrotic or infected tissue will expedite wound healing. There are numerous methods available but no consensus on which one is most effective for surgical wounds. OBJECTIVES: To determine the effect of different methods of debridement on the rate of debridement and healing of surgical wounds. SEARCH METHODS: In March 2013, for this third update, we searched the Cochrane Wounds Group Specialised Register; the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library); Ovid MEDLINE; Ovid MEDLINE (In-Process & Other Non-Indexed Citations); Ovid EMBASE; and EBSCO CINAHL. SELECTION CRITERIA: We included randomised controlled trials (RCTs) with outcomes including at least one of the following: time to complete debridement or time to complete healing. DATA COLLECTION AND ANALYSIS: Two review authors independently reviewed the abstracts and titles obtained from the search, extracted data independently using a standardised extraction sheet and independently assessed methodological quality. One review author was involved in all stages of the data collection and extraction process, thus ensuring continuity. MAIN RESULTS: Five RCTs (159 participants) were eligible for inclusion; all compared treatments for infected surgical wounds and reported time required to achieve a clean wound bed (complete debridement). One trial compared an enzymatic agent (streptokinase/streptodornase) with saline-soaked dressings. Four trials compared the effectiveness of dextranomer beads or paste with other products (different comparator in each trial) to achieve complete debridement. Meta-analysis was not possible due to the unique comparisons within each trial. One trial reported that dextranomer achieved a clean wound bed significantly more quickly than Eusol, and one trial comparing enzymatic debridement with saline-soaked dressings reported that the enzyme-treated wounds were cleaned more quickly. However, methodological quality was poor in these two trials. AUTHORS' CONCLUSIONS: There is a lack of large, high-quality published RCTs evaluating debridement per se, or comparing different methods of debridement for surgical wounds, to guide clinical decision-making.

[Conservative treatment of subcapsular hematoma in preeclampsia and HELLP syndrome: a case report]. / Tratamiento conservador del hematoma hepático subcapsular en pacientes con preeclampsia y síndrome de HELLP coexistentes: reporte de caso y revisión bibliográfica.

BACKGROUND: Liver hematoma is a rare and serious complication of pregnancy associated with preeclampsia-eclampsia and HELLP syndrome. CASE REPORT: 27 years old patient with two pregnancies, first pregnancy with eclampsia, admitted with 36.5 weeks of gestation, blood pressure of 140-100 mmHg, epigastric pain, shoulder pain without peritoneal irritation and increased tendon reflexes. The requested preeclamptic profile supports the diagnosis of severe preeclampsia and HELLP syndrome. It was decided to terminate the pregnancy by abdominal route. Male product was obtained alive, 2,060 g, Apgar 8/9, gestational age of 38.2 weeks Capurro. A review did not report liver parenchymal. The evolution during mediate puerperium was torpid, the patient presented epigastric pain and shoulder pain, and there was a rise in transaminases (AST 687 U/L, ALT 813 U/L), progressive thrombocytopenia (113, 103/ pL), decreased hemoglobin, proteinuria and hypovolemic shock. Abdominal CT scan was requested, and it confirmed a heterogeneous liver image (117 x 85 x 104 mm) with a volume of 694 cc, suggesting hepatic hematoma. Serialized control of abdominal CT indicated liver hematoma resorption after 25 days of hospitalization. Seven days after discharge transaminase levels were normal. CONCLUSION: To consider in the diagnosis of preeclampsia and HELLP syndrome the likelihood of liver hematoma as an acute complication; early treatment improves the prognosis.

Debridement for surgical wounds.

BACKGROUND: Surgical wounds that become infected are often debrided because clinicians believe that removal of this necrotic or infected tissue will expedite wound healing. There are numerous methods available but no consensus on which one is most effective for surgical wounds. OBJECTIVES: To determine the effect of different methods of debridement on the rate of debridement and healing of surgical wounds. SEARCH STRATEGY: For this second update we searched the Cochrane Wounds Group Specialised Register (searched 13 April 2011); the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2011, Issue 1); Ovid MEDLINE (2007 to March Week 5 2011); Ovid MEDLINE (In-Process & Other Non-Indexed Citations, April 11, 2011); Ovid EMBASE (2007 to 2011 Week 14); and EBSCO CINAHL (2007 to 8 April 2011). SELECTION CRITERIA: We included randomised controlled trials (RCTs) with outcomes including at least one of the following: time to complete debridement or time to complete healing. DATA COLLECTION AND ANALYSIS: Two review authors independently reviewed the abstracts and titles obtained from the search, extracted data independently using a standardised extraction sheet and independently assessed methodological quality. One review author was involved in all stages of the data collection and extraction process, thus ensuring continuity. MAIN RESULTS: Five RCTs (159 participants) were eligible for inclusion; all compared treatments for infected surgical wounds and reported time required to achieve a clean wound bed (complete debridement). One trial compared an enzymatic agent (streptokinase/streptodornase) with saline-soaked dressings. Four trials compared the effectiveness of dextranomer beads or paste with other products (different comparator in each trial) to achieve complete debridement. Meta-analysis was not possible due to the unique comparisons within each trial. One trial reported that dextranomer achieved a clean wound bed significantly more quickly than Eusol, and one trial comparing enzymatic debridement with saline-soaked dressings reported that the enzyme-treated wounds were cleaned more quickly. However, methodological quality was poor in these two trials. AUTHORS' CONCLUSIONS: There is a lack of large, high-quality published RCTs evaluating debridement per se, or comparing different methods of debridement for surgical wounds, to guide clinical decision-making.

Association of HL-A 5 and immune responsiveness in vitro to streptococcal antigens.

Lymphocytes, from randomly selected individuals having normal immune function, when incubated in vitro with varying concentrations of streptococcal antigens, responded in three ways: (a) response over the entire antigen concentration range, i.e., responders; (b) low response to only the highest antigen concentrations; and (c) no response at any antigen concentration. Frequency distribution analysis of these groups indicated that a significant association occurred between the ability to respond and HL-A 5.

Requirement of precommitted cells as targets for the augmentation of lymphocyte proliferation by leukocyte dialysates.

After our initial report tha leukocyte dialysates containing transfer factor augment the thymidine incorporation of antigen-stimulated lymphocytes, we have adapted the system to microleukocyte cultures. This modification permits both (a) the simultaneous assay of a single dialysate on the cells of multiple individuals, and (b) the assay of multiple dialysates on the cells of a single individual. The data thus secured, demonstrate that dialysates from both skin-test-positive and -negative donors produced similar degrees of augmentation whether the data are expressed as an arithmetic difference or as a ratio. When expressed as an arithmetic difference, the amount of augmentation is increased in proportion to the level of thymidine incorporation of the assay cells when they were stimulated by antigen alone. When expressed as a ratio, however, the degree of augmentation is independent of the response of the assay cells. An analysis of the ability of dialysates to engage previously uncommitted lymphocytes and thus to augment thymidine incorporation, revealed that precommitted cells were required. In these experiments, antigen-reactive cells were deleted from populations of peripheral blood lymphocytes by incubation with purified protein derivative of tuberculin, diphtheria toxoid, or streptokinase-streptodornase in the presence of [3H]thymidine of high specific activity. This deletion depressed or abolished the effect of dialysate on the residual population when it was recultured with the same antigen, but the effect on the response of the remaining lymphocytes to other antigens was unaltered. In this study, leukocyte dialysate appeared to augment nonspecifically the thymidine incorporation of an antigen-specific precommitted clone of lymphocytes. The relationship of these adjuvant effects on peripheral blood lymphocytes in vitro to the specific and nonspecific activities of transfer factor in vivo remains to be elucidated.

Cellular immunity in vitro. Clonal proliferation of antigen-stimulated lymphocytes.

When sensitive lymphocytes are cultured with the appropriate antigen, lymphoblasts appear after 24-48 hr of incubation and the number of these increases steadily from the 2nd to the 6th or 7th day. Our problem was to discover, at a cellular level, how this increase takes place; whether it is a massive response of many cells, stepwise recruitment of cells into the lymphoblast class, or simply repeated division of a few cells to form clones. In these experiments lymphocytes were incubated with antigen in culture tubes for 2-4 days and then a few cells, usually less than 200, were transferred to special microchambers for further culture. In these microchambers the cells could be viewed continually with a microscope and their fate recorded over the next 3-5 days by time-lapse cinemicrography. Examination of the film produced in this way showed that lymphoblasts divided and redivided to produce clones of 64 cells or more. It was possible to measure generation times from the film for 301 cells; the majority were between 8 and 13 hr but the range was 7.5-38.0 hr. There was no clear difference between generation times of human lymphocytes stimulated with tuberculin, streptokinase-streptodrnase, extract of the American pokeweed, or in the mixed leukocyte reaction. Similar times were also found for rat cells in the mixed leukocyte reaction. While these observations show that clonal proliferation does occur and could reasonably account for all the increase of lymphoblasts in lymphocyte cultures, the experiments, because of their design, do not exclude the possibility that other mechanisms such as recruitment may play a role as well, particularly during the first 48 hr after contact between sensitive cells and antigens.

Effect of normal and activated human macrophages on Toxoplasma gondii.

Human macrophages derived from in vitro culture of peripheral blood monocytes were studied under a variety of conditions to determine their microbicidal capacity for the obligate intracellular protozoan, Toxoplasma gondii. The effect of macrophages on intracellular Toxoplasma was evaluated morphologically by light and phase microscopy and by autoradiography. When macrophages from dye test (DT)-negative or DT-positive individuals were infected with Toxoplasma in the presence of normal human serum, the organisms were able to multiply intracellularly with resultant destruction of the monolayer. Once organisms were intracellular, the presence of antibody-containing serum in the medium did not alter this inability of the macrophages to kill Toxoplasma. However, when Toxoplasma were incubated in the presence of heat-inactivated DT-positive serum just before infection of the monolayers, the intracellular organisms were inhibited or killed by normal macrophages. Attempts were made to activate macrophages in vitro to kill Toxoplasma. Macrophages incubated in the presence of sensitized lymphocytes and Streptokinase-Streptodornase (SK-SD) or Toxoplasma lysate antigen (TLA) were found to kill Toxoplasma when compared to macrophages incubated in the presence of lymphocytes from DT-negative individuals and TLA or lymphocytes alone. Thus, in vitro induction of resistance (both specifically and nonspecifically) in human macrophages was accomplished by culturing these cells in the presence of specifically sensitized lymphocytes and antigen. These results suggest that, as in the mouse model, activated human macrophages have the ability to inhibit or kill intracellular Toxoplasma and that these cells may be important as effector cells in cell-mediated immunity (CMI) to toxoplasmosis in man.

Modulation of human lymphocyte function by C3a and C3a(70-77).

Human C3a and the synthetic octapeptide C3a (70-77), which retains the activities of an anaphylatoxin, inhibit in a concentration-dependent manner the generation of leukocyte inhibitory factor (LIF) activity by human mononuclear leukocytes and T lymphocytes cultured with the mitogens phytohemagglutinin (PHA) or concanavalin A (Con A) or the antigen streptokinase-streptodornase (SK-SD). The generation of LIF activity was inhibited by 50% by 10(-8) M C3a or C3a(70-77) with PHA or Con A as the stimulus, whereas a more than 10-fold higher concentration of C3a(70-77) than C3a was required to achieve the same level of suppression with SK-SD as the stimulus. Similar concentrations of C3a(70-77) inhibited to the same extent the migration of T lymphocytes stimulated by alpha-thioglycerol of Con A. Neither C3a nor C3a(70-77) altered significantly the uptake of [3H]thymidine by human mononuclear cells exposed to PHA, Con A, or SK-SD. The capacity of C3a(70-77)-Sepharose,m but not Sepharose alone, to adsorb or inactivate mononuclear leukocytes required for the generation of LIF activity established a direct interaction. Analysis of the lymphocytes in the effluent from C3a(70-77)-Sepharose columns, using monoclonal antibodies to surface antigens, showed a selective depletion of the helper/inducer population of lymphocytes. C3a might represent an important mediator of the functionally selective regulation of human T lymphocyte activities by the complement system.

Studies on mediator production by highly purified human T and B lymphocytes.

Highly purified populations of T and B lymphocytes obtained by affinity column separation were stimulated by antigen and their ability to produce two mediators, migration inhibitory factor (MIF) and lymphocyte mitogenic factor (LMF) was assessed. Both T- and B-cell populations made MIF; the production of MIF was antigen-specific using purified protein derivative of tuberculin, streptokinase-streptodornase, and Candida antigens. The MIF activity from both populations could not be attributed to antigen-antibody complexes as the inhibitory activity eluted from Sephadex G-100 columns in the same region corresponding to mol wt 23,000 daltons. Further studies indicate that the T cells producing MIF are proliferating cells whereas the B cells producing this mediator are not. In contrast, LMF was made only by T cells and not B cells when these populations were stimulated by antigen. The LMF induced the [(3)H]thymidine incorporation into both T and B cells obtained from donors lacking sensitivity to the antigens used to elicit the factor. Chromatographic studies indicate that LMF eluted from Sephadex G-100 in a fraction of mol wt 23,000 daltons where MIF is also found; however, since B cells produce MIF but not LMF, these two factors appear to be distinct from one another. Some of the implications of these findings are discussed. The explanation for the production or lack of production of MIF by lymphocytes obtained from patients with immunodeficiency disorders requires reinterpretation.

Specificity of antigen recognition by human lymphocytes in vitro.

Delayed hypersensitivity reactions in vivo are exquisitely specific, in terms of both a lack of response after induction of tolerance and a response after sensitization. These studies in vitro demonstrate that this specificity, at least at the level of antigen recognition, is probably derived from different populations of cells, each responding to different antigens.

A comparison between ((3)H)-thymidine incorporation and isothermal microcalorimetry for the assessment of antigen-induced lymphocyte proliferation.

Lymphocyte transformation tests (LTT) are time-consuming radioactive assays used in the clinic for the determination of allergic drug reactions and extensively in basic immunological research. In the present study we propose an alternative method in the monitoring of T-cell responses by isothermal microcalorimetric (IMC) measurements of overall cellular heat production as a function of time. For mitogen-induced lymphocyte proliferation, we analyzed a concentration dependent effect of phytohemaglutinin (PHA) and both tests showed a good correlation. This was also the case for specific antigenic stimulation with Varidase(R) or tetanus toxoid. On the other hand, antigen-induced lymphocyte proliferation analyzed by pre and post influenza vaccine (Inflexal(R) V) samples, showed no such correlation. Our study suggests that IMC measurements, despite the advantages of simplicity, on-line recording of metabolic activity and no use of radioactivity, may be limited to monitoring mitogen-induced lymphocyte proliferation.

Suppressor function of peripheral blood mononuclear cells in normal individuals and in patients with systemic lupus erythematosus.

Normal peripheral blood mononuclear cells demonstrated increased DNA synthesis and secretion of newly synthesized protein when suboptimal concentrations of Concanavalin A (Con A) were added to the cultures after 24-h incubation in vitro. Cells stimulated by Con A, 1 mug/ml, after 24-h incubation demonstrated 3.0 times more tritiated thymidine incorporation, and 4.4 times more 14C-amino acid incorporation into newly synthesized secreted protein, than cells stimulated at 0 h (P less than 0.001). The acquisition of increased responsiveness was not abrogated by washing and resuspending the cells in fresh medium. Since the increased responsiveness could be inhibited by the addition to the cultures of small numbers of cells previously activated by Con A it is suggested that the enhanced reactivity acquired in culture represents the loss of a subpopulation of suppressor cells that modulate the T-lymphocyte response. Cells from nine patients with active, untreated systemic lupus erythematosus demonstrated normal responses to optimal concentrations of Con A added at 0 h, but an impaired response to Con A, 1 mug/ml. When these cells were incubated for 24 h, a significant increased response to Con A was not observed. This observation suggests that patients with active SLE lack circulating suppressor cells. When seven SLE patients were again studied after corticosteroid therapy had led to clinical improvement, the response to Con A, 1 mug/ml, added after 24-h incubation was similar to that observed in normal controls, suggesting that suppressor function in SLE returns as disease activity declines.

Depression of the lymphocyte transformation response to microbial antigens and to phytohemagglutinin during pregnancy.

Lymphocyte transformation (LT) responses to Chlamydia trachomatis, to four other microbial antigens, and to phytohemagglutinin (PHA) were studied in 201 women during pregnancy and/or 3-18 wk postpartum. The LT responses to all stimulants tested were significantly depressed during pregnancy when compared with postpartum LT responses. This difference occurred whether LT assays were performed in autologous or pooled heterologous plasma collected from nonpregnant donors. Among women studied in the third trimester and again postpartum, the autologous LT stimulation index (LTSI) rose from 1.7 to 3.4 (P less than 0.001) with C. trachomatis elementary body antigen, from 3.7 to 7.9 (P less than 0.001) with Candida albicans cell wall extract, from 4.5 to 7.8 (P = 0.008) with streptokinase-streptodornase, from 1.7 to 3.0 (P = 0.007) with fluid tetanus toxoid, from 1.7 to 2.8 (P = 0.046) with mumps virus skin test antigen, from 35.5 to 87.0 (P less than 0.001) with PHA (2 micrograms/ml), and from 107.2 to 181.9 (P = 0.007) with PHA (10 micrograms/ml). LT responses to C. trachomatis were compared in 52 pregnant women and 58 nonpregnant women; all the women had C. trachomatis isolated at the time of LT assay. Using either plasma supplement, the mean LTSI with C. trachomatis antigen was significantly higher in nonpregnant women than in pregnant women, regardless of trimester (P less than 0.001). Among 12 women who were serially tested and remained culture positive for C. trachomatis throughout pregnancy and the postpartum period, the mean autologous LTSI rose from 1.9 in the third trimester to 7.8 postpartum (P = 0.0004). These data are the first to show that the immune response to an ongoing bacterial infection is depressed during pregnancy and to definitively document the depressed LT responses during human pregnancy.

Purification and further characterization of human mononuclear cell histamine-releasing factor.

We have purified and further characterized a histamine releasing factor (HRF) derived from human mononuclear cells using gel-filtration HPLC, reverse-phase HPLC, anion exchange chromatography, and elution from SDS gels after electrophoresis. Considerable heterogeneity is seen, far exceeding that published in prior reports. Gel filtration HPLC yielded a major peak at molecular weight 30,000 and minor peaks at 50,000 and 12,000. Reverse-phase HPLC gave one major fraction in the void volume and an eluted peak at 50-60% acetonitrile. Accell QMA anion exchange HPLC revealed three peaks of activity; one in the void volume similar to that published previously using QAE-Sephadex, and peaks that eluted at 0.5 and 0.8 M ammonium acetate, respectively. Electroelution following SDS-PAGE yielded peaks at MW 12,000 and 15-17,000 plus variable peaks at 25-27,000, 31-34,000, and 80-90,000 D. Using a combination of the aforementioned procedures, we have purified molecular species of HRF at 41,000 and 17,000 D to apparent homogeneity, as judged by SDS PAGE and autoradiography. Since human interleukin 3 and granulocyte-macrophage colony-stimulating factor possess histamine-releasing capability, it is clear that multiple cytokines can share this activity. However, the major HRF we isolate from human mononuclear cells appears, thus far, to be unique.

The effect of in vivo hydrocortisone on subpopulations of human lymphocytes.

This study was designed to determine the effect of in vivo hydrocortisone on subpopulations of lymphoid cells in normal humans. Subjects received a single intravenous dose of either 100 mg or 400 mg of hydrocortisone, and blood was drawn at hourly intervals for 6 h, and then again at 10 and 24 h after injection. Profound decreases in absolute numbers of circulating lymphocytes and monocytes occurred at 4-6 h after both 100 mg and 400 mg of hydrocortisone. Counts returned to normal by 24 h. The relative proportion of circulating thymus-derived lymphocytes as measured by the sheep red blood cell rosette assay decreased maximally by 4 h and returned to base line 24 h after hydrocortisone. There was a selective depletion of functional subpopulations of lymphocytes as represented by differential effects on in vitro stimulation with various mitogens and antigens. Phytohaemagglutinin response was relatively unaffected, while responses to concanavalin A were significantly diminished. Responses to pokeweed mitogen were unaffected by 100 mg of hydrocortisone, but greatly diminished by 400 mg of hydrocortisone. In vitro responses to the antigens streptokinase-streptodornase and tetanus toxoid were markedly diminished by in vivo hydrocortisone. Reconstitution of monocyte-depleted cultures with autologous monocytes partially corrected the diminished response to antigens. This transient selective depletion of monocytes and subsets of human lymphocytes by a single dose of hydrocortisone is most compatible with a redistribution of these cells out of the circulation into other body compartments.

Impaired lymphocyte transformation in intestinal lymphangiectasia: evidence for at least two functionally distinct lymphocyte populations in man.

Intestinal lymphangiectasia is a disease characterized by hypoproteinemia and edema resulting from protein-losing gastroenteropathy secondary to abnormal intestinal lymphatics. Immunologic abnormalities associated with this disease include hypogammaglobulinemia, lymphocytopenia, skin anergy, and impaired allograft rejection. In the present study, the in vitro blastogenic transformation of lymphocytes from 12 patients with intestinal lymphangiectasia was assessed in order to gain insight into the mechanism of the cellular immune defect in this disease. Peripheral blood lymphocytes from patients with intestinal lymphagiectasia showed impaired in vitro transformation to nonspecific mitogens, specific antigens, and allogeneic cells when compared to equal numbers of cells from normal individuals. Patients with the most deficient in vitro reactivity tended to have the lowest serum albumin concentration and the lowest absolute lymphocyte count. Lymphocytes obtained from chylous effusions in each of the four patients studied transformed more vigorously than peripheral blood cells from the same patients. These results may be explained by the loss of recirculating, long-lived lymphocytes into the gastrointestinal tract, resulting in a relative depletion of the population of lymphocytes necessary for in vitro blast transformation. This disease thus represents a clinical analogue of animals with experimental thoracic duct drainage, and provides evidence for the existence, in man, of two functionally distinct lymphocyte populations. In addition, these findings establish a new mechanism of impaired delayed hypersensitivity and defective in vitro lymphocyte transformation, i.e. the gastrointestinal loss and consequent depletion of the long-lived, recirculating population of lymphocytes from the peripheral lymphocyte pool.

Production of antibodies specific for Fc, Fab', and streptokinase-streptodornase in vitro by peripheral blood cells from patients with rheumatoid arthritis and normal donors. Identification of immune complexes in culture supernatants containing hidden antibodies reactive with Fab' fragments of immunoglobulin G.

To study antibody (Ab) biosynthesis in rheumatoid arthritis (RA), the immunoglobulin (Ig)M anti-Fc, anti-Fab', and antistreptokinase-streptodornase (SKSD) produced by peripheral blood lymphocytes (PBL) were measured at intervals from 1 to 19 d in culture. PBL from 17 seropositive patients with active RA and 30 age-matched controls were evaluated. Within the first 24 h, PBL from six of eight patients released >30 ng IgM anti-Fc, even in the absence of pokeweed mitogen (PWM). This early release of Ab was blocked by cycloheximide. With or without PWM, PBL from normal donors did not release IgM anti-Fc until after 3-5 d in vitro. By day 9, unstimulated PBL from seven patients made > 100 ng IgM anti-Fc. Un-stimulated PBL from normals never made >95 ng of this Ab. When PWM was added, PBL from normal donors released as much IgM anti-Fc as was found in RA donor cultures. Paradoxically, addition of PWM to PBL of RA patients suppressed release of IgM anti-Fc in 4 of 17 cases to levels significantly below those found in unstimulated cultures of the same cells. Without PWM, PBL from RA donors frequently failed to make IgM anti-SKSD (P < 0.05 compared with normal donors' cells). With PWM, the quantities of IgM anti-SKSD released were comparable. Fluctuations in IgM anti-Fab' levels during the life-time of these cultures were sufficient to suggest that these Ab may be taken up in immune complexes. This hypothesis was verified by acidifying (pH 3.1) culture supernatants to which (125)I-Fab' had been added previously. The samples were then neutralized (pH 7.6) and 12% polyethylene glycol was added to separate free from antibody-bound (125)I-Fab'. This procedure increased the quantity of (125)I-Fab' precipitated by > 10-fold in some cases. These studies suggest that there are a variety of abnormalities in Ab biosynthesis in RA. These include spontaneous synthesis of comparatively large quantities of IgM anti-Fc, relatively suppressed release of IgM anti-SKSD, and a paradoxical reduction, in some cases, in the biosynthesis of IgM anti-Fc after addition of PWM.

Cytotoxicity of human macrophages for tumor cells. Enhancement by human lymphocyte mediators.

Human macrophages, derived from peripheral blood monocytes, acquire enhanced cytotoxicity for human target cells after incubation in mediator-rich supernates from antigen-stimulated lymphocytes. Maximum cytotoxicity was observed after 24-h incubation in mediators. In comparison to normal macrophages, mediator-activated macrophages were cytotoxic to five of the six malignant cell lines tested but had no effect on five nonmalignant cell lines. In 20 experiments with one target (SK-BR-3), mean cytotoxicity was 23 +/- 2.7% and with another target (MA-160), was 29 +/- 3.4%. Macrophages became cytotoxic after 8-h incubation with mediators and the enhanced cytotoxicity persisted for at least 40 h after the lymphocyte mediators were removed. These findings are consistent with the hypothesis that macrophages, activated by antigen-induced lymphocyte mediators, can contribute to the host resistance to tumor growth in man.

Degradation of human fibrinogen by plasms alpha2-macroglobulin-enzyme complexes.

This study demonstrates that human plasma alpha(2)-macroglobulin preparations possess an enzymic activity that degrades fibrinogen, resulting in the formation of products whose structure resembles that of circulating fibrinogen catabolites. The sequence of degradation is similar to that observed in plasmin-catalyzed digests, in that Aalpha-chain fragmentation precedes that of Bbeta-chain. The addition of plasminogen activators to plasma induced an increase in the N-alpha-tosyl-l-arginine methyl ester HCl esterase and fibrinogenolytic activity associated with alpha(2)-macroglobulin purified from this plasma, indicating that the enzymic activity of the complex was preserved and could be increased in the presence of other plasma enzyme inhibitors. Immunochemical studies demonstrated that an alpha(2)-macroglobulin-plasmin complex had formed in urokinase-treated plasma. This alpha(2)-macroglobulin preparation manifested an esterolytic profile like that of a complex prepared from plasmin and purified alpha(2)-macroglobulin. After complex formation with alpha(2)-macroglobulin in plasma, plasmin retained less than 0.1% of its fibrinogenolytic activity. That plasmin expressed its activity while bound to alpha(2)-macroglobulin was suggested by immunoprecipitation of this activity with alpha(2)-macroglobulin antibody and by the demonstration that pancreatic trypsin inhibitor did not effectively inhibit its fibrinogenolytic or esterolytic activity. These results raise the possibility that, in addition to its activity as a major plasma proteolytic enzyme inhibitor, alpha(2)-macroglobulin may modulate enzyme-substrate interactions, such as those resulting in the formation of circulating fibrinogen catabolites, by providing a mechanism for the preservation and protection of a portion of the enzymic activity in the presence of other circulating inhibitors.