Modulation of IgG effector functions by a monovalent fragment of staphylococcal protein A.

The monovalent V-1 fragment of protein A (fSpA) with a mol. wt of 13,000 obtained from an u.v. mutant of Staphylococcus aureus Cowan I strain was proved to be able to modulate significantly some of the effector functions of IgG, such as complement fixation, catabolism, attachment to Fc receptors and antibody-dependent cell-mediated cytotoxicity. Moreover fSpA-like protein A obtained from the A676 strain is mitogenic and enhances NK activity of human peripheral lymphocytes. The efficiency of fSpA was found to be lower than that of protein A with regard to its ability to inhibit complement fixation, EA rosette formation and antibody-dependent cell-mediated cytotoxicity. Both protein A and fSpA had the same efficiency in activation of the complement system after reaction with human or guinea pig IgG, and in increasing the IgG catabolism. Unlike fSpA the monovalent B fragment of protein A (with mol. wt of 7000) was not able to inhibit EA rosette formation and antibody-dependent cell-mediated cytotoxicity. The results recommend fSpA, substituting for protein A, as a molecular probe for the investigation of IgG antibody and lymphocyte effector functions.

Correlation between the triggering of proliferation and the potentiation of NK activity induced by protein A in human lymphocytes.

Various preparations of Staphylococci protein A (SpA) obtained either from strain Cowan-1 or strain A676 and two SpA low molecular fragments (AB and B) were tested for their mitogenic and NK-stimulating activity on human peripheral blood lymphocytes. Though all 4 preparations of SpA and both of its fragments were able to react with human IgG, apparently with both Fab and Fc sites of IgG, only the SpA preparations derived from A676 strain triggered the proliferation and potentiated the NK-activity of treated cells. The parallelism between the mitogenic and NK-stimulating activities of SpA suggests that both activities might be under the control of the same portion of the SpA molecule located in the non-immunoglobulin binding region of the molecule.

Aging influence on the E-rosette forming cells, spontaneous cell mediated cytotoxicity, and antibody dependent cellular cytotoxicity in humans.

In 131 adults and 107 aging healthy subjects of both sexes, the percentage of the T lymphocytes in peripheral blood and the cytotoxic activities of NK and K cells, were determined. No age-related differences have been found in the number of peripheral blood lymphocytes, the binding of NK cells to the K562 targets, the percentage of E rosette forming cells and the ADCC level. An increased proportion of T suppressor lymphocytes in elderly subjects and a large inter- and intra-individual variation of NK cells activity in both adult and aged subjects were found.

Natural cell-mediated cytotoxicity in Ehrlich ascites tumor-bearing mice.

The level of NCMC (natural cell-mediated cytotoxicity) in the spleen of Ehrlich ascites tumor-bearing mice at every 5 days after i. p. tumor transplantation, and the effect of cell-free ascitic fluid or serum of tumor bearers on natural cytotoxicity in vitro, were assessed. Spleen lymphocytes were used as effectors and 51Cr-labeled YAC1 cells as targets at the effector: target ratio of 200 : 1. In tumor bearers, the NCMC increased in the first days after i. p. inoculation of 1 X 10(6) Ehrlich cells with a maximum peak on day 5. Thereafter, the activity decreased continuously, on day 20 representing about half of normal value. Ascitic fluid and serum of tumor bearing mice significantly depressed the NCMC activity exerted in vitro by spleen lymphocytes of normal mice.

In vivo and in vitro effect of Cantastim, an immunomodulatory agent extracted from a highly pathogenic Pseudomonas aeruginosa strain.

Cantastim is a biological product consisting of distinct molecules of phospholipids obtained from a peculiar Pseudomonas aeruginosa strain. It enhanced the IgM and especially the IgG responses of mice pretreated with Cantastim and then intraperitoneally inoculated with T-dependent antigens such as sheep erythrocytes; restored cellular immunocompetence as evidenced by an increase in the in vitro proliferative responses to T-cell mitogens and allogeneic stimuli of murine splenocytes from in vivo pretreated mice activated NK cytotoxicity, and inhibited in vivo growth of the Ehrlich ascites tumor. In vitro it stimulated the mitogenesis of mouse lymphocytes but did not exert such stimulatory effect on guinea pig or human lymphocytes. All these findings allow to define Cantastim as an immunomodulating agent influencing mainly the cell-mediated immune response.

A therapeutical outlook in "infectious" bronchial asthma.

Chronic bronchial asthma seems to be generated by abnormal functions of PMNs and eosinophils stimulated by local bacterial flora. Once stimulated, they release uncontrolled excessive amounts of ROI and various soluble mediators. These are the expression of disorders in the synthesis of SOD, catalase and other enzymes by PMNs and an excessive production of the so-called anti-inflammatory Th2 type cytokines, mainly IL-4 and IL-5 to the detriment of IFN gamma and other Th1 type cytokines. Treatment consisting of autovaccines obtained from local flora associated with immunomodulators instead of antibiotics or glucocorticoids proved to be beneficial for patients.

DNA cell cycle analysis of K562 and Ehrlich tumor cells by flow cytometry.

Monitoring cell activation processes and cell cycle progression in cells of various ontogenetic and phylogenetic origins is one of the base challenges in cellular biology. Flow cytometric analysis by DNA histograms show the cell cycle distribution and indicate the S-phase fractions of cell populations, as well as those cells in Go/G1 and G2/M phases. Human and murine normal diploid cells, K562 tumor cells (human origin) and Ehrlich cells (murine origin) were analysed using a propidium iodide staining procedure for flow cytometry. Our preliminary results suggested that this method may be a useful tool to detect cell populations with quantitative changes in DNA content, and to estimate cell fractions.

Procain and diethylaminoethanol influence on the release of free oxygen radicals by polymorphonuclear leukocytes, in rabbits and humans.

The investigations were conducted on 3 groups of New Zealand rabbits: 1) controls; 2) injected with procain, i.m. 15 mg/kg body weight, daily, for 30 days; 3) injected with diethylaminoethanol (DEAE), 15 mg/kg body weight, daily, for 35 days. The study was made also on human leukocytes, isolated from the peripheral blood of 10 clinically healthy subjects (adults), procain and DEAE action being investigated in vitro. The free oxygen radicals (FOR) released by PMN leukocytes were evaluated by chemiluminescence, in vitro. Addition of procain or DEAE had no effect on the release of FOR by PMN leukocytes of control rabbits. In the experiment made on rabbits treated with procain or DEAE, the release of FOR by PMN leukocytes was much more reduced, as compared to controls. In the rabbits treated with procain, the intensity of the emitted light was 2.27 mV, in those treated with DEAE, 3.46 mV, while in the controls, the mean value was 6.74 mV. In the in vitro experiments performed on human PMN cells stimulated with opsonized zymosan (OZ), addition of procain or DEAE had an inhibiting effect on the FOR release. As compared to control, the means of the FOR values decreased from 59 to 41.2 mV in case of procain addition and from 67.7 to 50 mV in case of DEAE addition. The fact that the inflammation is associated with accumulation of free radicals, suggests the opportunity to test these substances, especially DEAE, as antioxidant agents.

The levels of neutrophils oxidative burst in rheumatic disorders.

We investigated the participation of the polymorphonuclear leukocytes (PMNs) as mediators of tissue destruction in 2 variants of rheumatic disorders that affect primarily the joints: the rheumatoid arthritis (RA) and the osteoarthritis (OA). We noted significant differences in the number of PMNs present at the joint level, which was low in OA and high in RA. The unstimulated and in vitro stimulated with opsonized zymozan (OZ) or ConA PMNs from the peripheral blood of these two groups of patients released a normal level of oxygen free radicals (OFR). On the contrary, the neutrophils isolated from the synovial fluid (SF) of RA patients showed a distinct feature: the unstimulated cells released high amounts of OFR, while those in vitro stimulated with OZ or ConA presented a low level of respiratory burst. In OA both in vitro unstimulated and stimulated with OZ or ConA neutrophils isolated from the SF had diminished values of OFR released. We assume that the chemiluminescence assay could detect distinct differences of the PMNs implications in RA and OA injuries and may be a laboratory test in the differential diagnosis of these rheumatic disorders.

Action of some proteic and carbohydrate components of Symphytum officinale upon normal and neoplastic cells.

The crude watery extract of Symphytum officinale and certain proteic and carbohydrate components isolated from it were studied for their effect upon the in vivo and in vitro proliferation of Ehrlich ascites cells, EL-4 cell line and of human T lymphocytes and upon the respiratory burst of human PMN granulocytes stimulated via Fc receptors. The results indicate that the crude extract and its proteic fraction stimulate the in vivo proliferation of the studied neoplastic cells and exert an antimitotic effect on human T lymphocytes in vitro stimulated with PHA. The vegetal preparations have remarkable effects on the respiratory burst of the granulocytes non-stimulated and stimulated via Fc receptors. The data underline the necessity to study thoroughly the effects of different phyto compounds through both pharmacological and immunological methods.

Flow cytometric determination of Enterococcus spp. susceptibility to four antimicrobial agents.

Two Enterococcus strains (E. faecalis and E. faecium) isolated from 2 patients in an intensive care unit (blood and drain, respectively) were analyzed for susceptibility to 4 antibiotics (penicillin, vancomycin, gentamicin, streptomycin) by agar dilution standard method (MICs), time-kill and flow cytometry. We compared the data from classical methods of antibiotic susceptibility detection, that are compulsory 24 hrs long and flow cytometry results at 5 and 24 hrs cultivation. The results from both classical and flow cytometric analyses were highly cogent and revealed the fact that flow cytometry is very useful in early diagnosis of bacterial resistance to antibiotics.

The influence of some vegetable extracts on the in vitro adherence of mouse and human lymphocytes to nylon fibers.

We found that the total watery extracts obtained from roots of various plants such as Symphytum officinale, Phytolacca americana etc, precipitate human glycoproteins, agglutinate sheep red blood cells (SRBC) and stimulate lymphocyte adherence to nylon fibers. Five out of seven extracts precipitated human gammaglobulins and one of seven obviously agglutinate SRBC. If these cells were pretreated with rabbit antibodies against SRBC, all extracts agglutinated the cells at various degrees of intensity, the most active being Phytolacca americana. The adherence of mouse but not human lymphocytes to nylon fibers was stimulated by extracts of Symphytum officinale and Phytolacca americana. This process was neither stimulated nor inhibited by Mannose (Man), Galactose (Gal), Glucose (Glc), N-acethyl Galactose (GalNAc) and N-acethyl Glucose (Glc-NAc). These biological effects of the plant extracts could be the expression of a lectin-like ability to bind various sugars other than those mentioned. The results suggest the possibility of using different extracts as means to point out the presence in serum or at the cellular level of some carbohydrates influencing the cellular adhesion, phenomenon which plays an important role in the functions of hematopoietic cells.

Modulation of CD14 expression on human granulocytes by a bacterial product "Cantastim" derived from Pseudomonas aeruginosa.

Pathophysiological effects of infection with Gram-negative bacteria depend on recognition by host cells of LPS, a major component of the outer membrane of these microorganisms. CD14 is an immunological marker and also the receptor for LPS on phagocytic cells. The aim of the present study was to investigate the capacity of a bacterial immunomodulator, named Cantastim, which is used as a therapeutical agent in neoplasic diseases and chronic infections, to modulate the CD14 expression on human granulocytes. Our preliminary results showed that Cantastim can not influence the expression of CD14 on granulocytes obtained from healthy donors, but on the other hand Cantastim was able in vitro and in vivo to down-regulate the expression of this marker on granulocytes from a patient with neoplasic disease.

Analysis by flow cytometry of the subpopulations of lymphocytes from the peripheral blood of procain or diethylaminoethanol treated rabbits.

The authors worked on 3 groups of 8 male rabbits, New Zealand race: 1) controls; 2) procain injected i.m., 15 mg/kg body weight, daily, for 30 days; 3) i.m. injected with diethylaminoethanol (DEAE), 15 mg/kg body weight, daily, for 35 days. The expression of the MHC I, MHC II, CD43, CD4 and IgM antigenic markers on the plasmatic membrane of the lymphocytes was studied using flow cytometry and monoclonal antibodies. Procain or DEAE treatment reduced the percentage of lymphocytes expressing I MHC, from 99.06 in the control group, to 94.51 in procain group and to 96.91 in the DEAE group. The intensity of expression of MHC complexes of class II decreases from 160.94 in the control group, to 107.21 in the procain group and to 104.05 in the DEAE group. No significant differences were noticed between the three groups of rabbits concerning the rate of lymphocytes that have on their surface expressed markers for CD43 (lymphocytes T), CD4 (Th), or IgM (lymphocytes B). Lymphocytosis induced in rabbits as a result of the DEAE treatment took place without a change in the proportions of lymphocyte subpopulations. The authors consider that owing to their capacity to reduce the expression of antigens MHC of class I and class II on the membrane of lymphocytes, procain and DEAE can have benefic effects in some autoimmune, autoaggression and inflammatory diseases.

The influence of synovial fluid on lymphocyte functions in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic recurrent and systemic inflammatory disease affecting around 1% of the population, that primarily involves the joints. In this study, we determined the Th1/Th2 lymphocytes ratio at the site of rheumatoid inflammation and the influence of the synovial fluid (SF) on the secretory and proliferative function in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC), obtained from patients with RA. Our results showed significant differences concerning the mononuclear cells and the CD4/CD8 ratio in synovial fluid and peripheral blood of patients. In SF prevailed Th1 cells, while in peripheral blood we found another cytokine profile of T lymphocytes. Also synovial fluid lymphocytes had a low PHA-stimulated blastogenic response. Patients plasma and synovial fluid showed an inhibitory effect on prolipheration indexes.

The migratory and phagocytic activity of polymorphonuclear leukocytes in rheumatoid arthritis and osteoarthritis patients.

Polymorphonuclear neutrophils (PMN) play a central role in the elimination of most extracellular pathogenic microorganisms and any impairment of their functions therefore predisposes to defect immune defence. We investigated the migratory and phagocytic functions of the PMNs isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The results suggest that in RA the number and the migratory but not phagocytic capacity of synovial fluid (SF) neutrophils were enhanced, while in OA they were significantly decreased in synovial fluid cells comparatively with peripheral blood (PB). The migratory function of both PB and SF cells from RA patients was increased comparatively with that of the cells from OA patients. We found the different abnormal functions in synovial fluid neutrophils from RA and OA patients. These results may help to elucidate the underlying mechanism which leads to severe joint destruction and different susceptibility to infectious diseases in patients with rheumatic disorders.

Evaluation of the safety of cantastim in normal humans.

CANTASTIM, a good immunomodulatory agent obtained from a highly pathogenic strain of Pseudomonas aeruginosa was inoculated during 12 weeks in 21 healthy individuals aged 18-45 years. The subjects were clinically and laboratory investigated for body weight, antibodies against DNA, collage, cardiolipin, number of blood cells, blood and urine enzymes etc. After this intensive therapy, all the studied parameters were found in normal range of values. The results are a proof that this immunomodulator is not antigenic and has not side effects.

Shift toward T lymphocytes with Th1 and Tc1 cytokine-secterion profile in the joints of patients with osteoarthritis.

Osteoarthritis (OA), although a heterogeneous disease, is generally believed by rheumatologists to be primarily a disease generated by biomechanical alterations. In order to determine the role of T cells, the pattern of T lymphocyte cytokines and to characterize the mononuclear cells from both peripheral blood (PB) and synovial fluid (SF) from patients with OA flow cytometry with a panel of monoclonal antibodies recognising CD4, CD8 and intracellular cytokines (IL2, IL4, IL10, gamma-IFN) was employed. The coexpression of CD4 and CD8 markers only on the SF T cells surface after in vitro stimulation by phorbol-miristate acetate and ionomicine, but not on PBL or in vitro unstimulated SFL was noted. The intracellular IFN-gamma was detected in enhanced levels in both CD4+ and CD8+ SF T cells, while the IL2 contents was not different in PB and SF samples. The Th2/Tc2 cytokines (IL4 and IL10) were detected in low amounts in both PBL and SFL. This study shows the role of some T cell populations and cytokines in the generation of joint destruction in osteoarthritis.

The cytochemical reaction of alpha-naphthyl-acetate esterase (ANAE) in peripheral blood lymphocytes of human cancer.

Peripheral blood lymphocytes from neoplasic patients have been investigated by cytochemical analyses of alpha-naphthyl-acetate-esterase (ANAE), marker of both cellular types and differentiation, maturation stages in relation with immunobiologic background of cancer. Our studies employed 32 cancerous human patients and compared them to 13 healthy people used as controls. The cytoenzymatic analysis was performed on whole blood samples, mononuclear cell concentrates (PBL) and rosetted ET and E45 preparations. ANAE dot like reaction, marker for mature T lymphocytes, considered by some authors to be specific to T helper lymphocytes, is present in a reduced number of lymphocytes, markedly below the control values. ANAE dot like positive lymphocytes reached average values of 22.5% (12-30%) in peripheral blood samples, 22% (6-32%) in mononuclear cell concentrate (PBL) and 21.3% (12-30%) in rosetted ET preparations, respectively. Comparing the cytoenzymatic data to those immunologically obtained with rosetted ET, we found out that the average values of ANAE dot like reacted lymphocytes, i.e. 22% (22.9-24.3%) were much lower than those of ET lymphocytes rosetted with sheep red blood cells, i.e. 60% (32-75%). The percentage of ANAE dot like reacted lymphocytes was also lower than that of T helper lymphocytes (40.2%). Therefore, there is a dissociation between ET rosetting capacity and the expression of ANAE dot like enzymatic reaction, the latter being more reduced than the former. Our results indicate that the deficiency present in ANAE dot like reaction is due to a constant important decrease of ANAE dot like positive lymphocytes, what suggests a functional deficiency of T helper lymphocytes in neoplasic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of products of plant and microbial origin on phagocytic function and on the release of oxygen free radicals by mouse peritoneal macrophages]. / Influenta unor produsi de origine vegetala sau microbiana asupra functiei fagocitare si eliberarii radicalilor liberi de oxigen de catre macrofagele peritoneale murine.

The effect of in vivo stimulation with an aqueous extract obtained from roots of Symphytum officinale and Cantastim on mouse peritoneal macrophages was investigated. The results obtained showed that these products initially activated the respiratory burst of the cells and later inhibited it, activating the synthesis of catalase, SOD etc. These data suggest that macrophages challenged by various ingested antigens destroy them initially through oxygen dependent mechanisms and later through enzymatic digestion in order to retain unimpaired their epitopes.