Inhibition of human monocyte spreading. An in vitro test for immunosuppressive potency of antihum lymphocyte globulin.

Monocytes from human peripheral blood, when incubated in vitro, spread onto the surface of the glass. Horse antihuman lymphocyte globulin (ALG) added to the incubation chamber inhibits the spreading, while normal horse globulin (NHG) does not. The inhibition depends on the concentration of ALG admixed to the mononuclear blood cells. Eleven coded samples of antihuman ALG were assayed for the ability to inhibit monocyte spreading. This potential was then compared to the in vivo immunosuppressive effect of the same samples determined by the prolongation of skin allograft survival in subhuman primates. It was found that the in vitro inhibitory acttivity correlated rather well with the in vivo immunosuppression, Therefore, the inhibition of monocyte spreading is proposed as an additional test system for the in vitro evaluation of the immunosuppressive potential of antihuman ALG.

Modulation of [Ca2+]i in freshly isolated mouse lymphocytes with in vivo priming.

Studied at the level of the individual cell, the pattern of [Ca2+]i mobilization of in vivo sensitized mouse lymphocytes by T-dependent antigen (KLH), challenged in vitro by Con A, PHA or anti-CD3epsilon mAb in different periods after immunization, was as follows. In the entire DLN lymphocyte population and in tested T cell subsets from immunized mice, baseline [Ca2+]i was significantly increased and cells were able to respond additionally to stimuli. In KLH-primed DLN lymphocytes, calcium mobilization in response to membrane receptor-dependent stimuli (anti-CD3epsilon, PHA, and ConA) was increased. Enhancement of Ca2+ mobilization is parallel with changed immunophenotype. These findings suggested that: (a) [Ca2+]i mobilization could correlate with lymphocyte behaviour during immunization and that mobilization clearly depended on kinetics of immune reaction; (b) the higher level of activity among sensitized lymphocytes was due to the increased number of specific B-cells (Ia(k+)) and gammadeltaTCR+ cells; (c) the quantitative measurement of [Ca2+]i could be an important biochemical parameter to study cellular reaction to a specific antigen.

The role of cytokines in MET-enkephalin-modulated nitric oxide release.

In the present study the in vitro and in vivo effect of Met-enkephalin (MENK) on nitric oxide (NO) release by mouse peritoneal macrophages was evaluated. While in vitro MENK was ineffective unless combined with suboptimal concentrations of recombinant murine interferon gamma, in vivo all the doses (2.5, 5 or 10 mg/kg bw) bimodaly modulated NO release. Only the stimulative (2.5 and 10 mg/kg bw) and not the suppressive (5 mg/kg bw) dose of MENK was opioid receptor-mediated as demonstrated by abolishing the effect by naloxone. The stimulative effect of the low (2.5 mg/kg bw) dose, that was observed only if MENK was injected p.m., was associated with the IL production and IFN gamma as demonstrated by abolishing the effect by specific antibodies. The data additionally support the idea that opioid-mediated responses might be to a large degree mediated by the release of cytokines.

Introduction of the gene amplification technique to decrease the risk of hepatitis C virus transmission by plasma products.

The viral safety of plasma-derived products with respect to hepatitis C virus (HCV) is assured by selection of donors, screening of individual donations for antibodies to HCV and the incorporation of effective viral inactivation-removal steps into manufacturing processes. As antibody screening of single donations is not sufficient to completely eliminate HCV RNA positive plasmas from plasma pools, testing for HCV RNA by gene amplification techniques may be necessary to identify positive donations. Using modern molecular biology techniques, we developed a specific, sensitive and reproducible method for routine PCR screening for HCV RNA in plasma pools.

Intrapleural application of natural IFN alpha in breast cancer patients with pleural carcinomatosis. Monitoring of immunotherapy by assaying serum interferon levels.

For resistant local recurrence, e.g. in breast cancer, or metastatic spread, local infiltration of IFN may be an interesting new approach. The aim of this study was to find out if intrapleurally administered interferon, in breast cancer patients with pleural carcinomatosis, can cause measurable serum concentrations and how soon after administration. Serum IFN concentrations were compared with those in the pleural fluid, and correlated with the presence of malignant cells in the pleural fluid. To uncover possible rhythmicity of serum interferon levels and its relationship to the timing of therapy, natural leukocyte interferon was administered intrapleurally at 10 a.m. Data on pharmacokinetics were obtained from blood samples drawn at -2, 0, 2, 8, 14, 22 and 46 h during the course of treatment. In contrast to our previous observations in healthy volunteers, levels of serum IFN before therapy had no circadian rhythmicity. Daily pharmacokinetic profile of individual patients on interferon therapy has shown that serum IFN peaks 8 h after intrapleurally administered IFN alpha. The peak depended on frequency and number of applied doses. During treatment with IFN alpha, malignant cells degenerated and finally disappeared from pleural fluid. At the same time reactive cells appeared. This effect is rather uniformly observed, but varies in degree. The number of patients is too small, however, to permit conclusions in regard to correlation of this clinical effect and the levels of serum IFN alpha.

gamma delta TCR+ intestinal intraepithelial lymphocytes (i-IEL) in reaction against intestinal nematode Trichinella spiralis.

To assess the gamma delta TCR T cells in the control of the timing of the mucosal response to enteric parasitic infections, we used C57BL mice, orally infected with 200 viable T. spiralis larvae. The small intestine, spleens and Peyer's patches (PP) were excised on 1, 4, 7, 14, 21 and 29 postinfection days (p.i.) for immunophenotyping and histological studies. Uninfected mice served as control. Characterization of isolated lymphocytes of C57BL control mice, confirmed that T cell immunophenotype differs in spleen, PP and i-IEL. Practically all i-IEL were CD3+ cells (83%). In addition, most of the i-IEL expressed Ly-2 (65%). Among the i-IEL, the level of gamma delta TCR+ cells was significantly higher (29%) than that found in spleen (3%) and PP (3%). The expression was high on CD3+ and Ly-2+ (26 and 21%, respectively) and low on L3T4+ i-IEL (< 1%). During T. spiralis infection alpha beta TCR+ CD3+, gamma delta TCR+ CD3+ and gamma delta TCR+ Ly-2+ i-IEL increased on day 4 and 7. However, infected mice displayed a reduction in i-IEL number from 14 to 29 p.i. day. At the same time the proportion of gamma delta TCR on spleen Ly-2+ and on PP CD3+ and Ly-2+ cells increased on 14 and 21 p.i. day. Adult worms were expelled from the gut by day 14. Thus, the kinetics of gamma delta TCR+ i-IEL, but not spleen and PP gamma delta TCR, corresponded to the kinetics of worm expulsion in C57BL mice. Most murine i-IEL of the gamma delta T cell lineage tend to be cytolytic when activated. We speculated that gamma delta T cells of i-IEL during the early stages of infection recognize and eliminate damaged epithelial cells generated by parasite antigens, simultaneously accelerating the worm expulsion.

Incidence of hepatitis C virus RNA in anti-HCV negative plasma pools in Croatia.

The risks of transmitting viral infection by blood and products derived from plasma have long been known and still remain an area of concern. Blood banks and transfusion centres are faced with the imminent introduction of nucleic acid amplification testing (NAT) of plasma pools as used by the plasma industry. In this paper, we show a part of our results of a validation study of an in-house method for routine polymerase chain reaction (PCR) screening for hepatitis C virus (HCV) RNA in plasma pools and the results of testing 2,718 anti-HCV negative plasma pools for the presence of HCV RNA. The European Committee for Proprietary Medical Products (CPMP) recommended that from 1 July 1999, only batches derived from plasma pools tested and found non-reactive for HCV RNA, using validated test methods of suitable sensitivity and specificity, should be batch released by authorities. The quality and efficiency of NAT detection of HCV RNA is among others influenced by the efficacy of RNA isolation, the primer selection and the use of control samples. Using modern molecular biology techniques (sensitive and specific in-house amplification methods for detection of HCV RNA and automated sequencing), we analysed samples of plasma pools from different Croatian transfusion centres. By detection of HCV RNA in an NIBSC working reagent (genotype 3) and a Pelispy HCV RNA run control (genotype 1) we determined a high reproducibility and sensitivity (below 100 International Units (IU)/ml) for our in-house method. By direct sequencing PCR cDNAs we proved the specificity of the test system and the possibility of determining the HCV genotype when the method was used for PCR screening of HCV RNA in single donations. Of 2,718 anti-HCV negative plasma pools we have found that 2.1$ were HCV RNA positive. Results of our investigation confirm the necessity of testing HCV RNA in plasma pools to further increase the safety of human plasma-derived drugs.

Interleukin 6 and its soluble receptor are elevated in aqueous humor of patients with uveitis.

PURPOSE: To determine the levels of interleukin 6 (IL-6) and its soluble receptor (sIL-6R) in the aqueous humor (AH) of patients with different uveitis entities. PATIENTS AND METHODS: AH and serum samples were collected from 35 patients (39 eyes) who underwent surgery for uveitis complications and from 10 controls (senile cataract). In the studied group, seven patients had HLA-B27(+) anterior uveitis, two had Fuchs' heterochromic iridocyclitis, 12 had chronic anterior uveitis of unknown etiology, and in the remaining 14 the causative agent was exogenous. The cytokine and receptor levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: In controls, the median IL-6 level in AH was higher than that in corresponding sera (40.4 pg/ml and 5.2 pg/ml, respectively). In contrast, sIL-6R showed an inverse relation: there were less sIL-6R in control AH than in control sera (378.9 pg/ml and 52749.0 pg/ml, respectively). The same qualitative relationship was observed in patients with uveitis. Quantitatively, in comparison to controls, elevated levels of IL-6 and sIL-6R were found in AH of patients with uveitis. As expected, the maximal IL-6 and sIL-6R values were observed in the patients with uveitis of exogenous etiology (1558.3 and 1326.2 pg/ml, respectively). sIL-6R was also significantly elevated in AH of patients with HLA-B27( +) anterior uveitis (p<0. 01). In all individuals under study, sIL-6R levels in AH samples were 2-10 times higher than IL-6 levels. In serum samples, sIL-6R level were 10000 times higher than corresponding IL-6 values. CONCLUSION: The results confirmed the role of IL-6 in intraocular inflammation and gave new information regarding the presence of its sR in normal and inflamed eyes. Low levels of sIL-6R in AH compared to those found in serum suggest the presence of active local regulatory mechanisms that require further investigation.

Immunophenotypization of cells involved in local immune response and serum antibodies in cephalosporin-treated mice.

The in vivo potency of cefodizime (HR 221), tiprotimod (a new synthetic thiazole derivative of HR 221, HBW 538) and cefotaxime to modulate the initiation of immune response in the draining lymph node (LN) after subcutaneous injection of SRBC was evaluated. The timing and sequence of events in the regional LN was investigated by immunophenotypization of node cells with monoclonal antibodies, and the systemic reaction was estimated as primary antibody response to SRBC. From the results it is possible to conclude that: (1) subcutaneous administration of a small dose (2.5-3.0 mg/kg) of cephalosporins, together with antigen, enhanced primary antibody production and persistence; (2) the increase in serum antibodies was preceded by a change in percentage of L3T4+ cells within the regional (popliteal) lymph node. In comparison to antigen alone, cephalosporins (during early immune response) increased the percentage of L3T4+ cells; (3) LN cellularity was strongly enhanced by cephalosporins; (4) cefotaxime influenced the kinetics of the cellularity and the L3T4/Lyt-2 index differently than cefodizime and HBW 538. HBW 538 had either a similar or stronger effect than cefodizime, as judged by the adjuvant effect on antibody production and the appearance of L3T4+ cells during the immunizing period.

[Juvenile papillomatosis of the larynx]. / Juvenilna papilomatoza larinksa.

Juvenile laryngeal papilloma is the most common childhood neoplasm of the larynx. It is most often multiple and is characterized by a strong tendency to recur. Seventy-eight children with primary juvenile laryngeal papillomatosis (JLP) were treated at the ENT Department of the Zagreb University School of Medicine in the last 20 years and in the last 6 years interferon has been introduced into the therapy. Causes of the recurrence and the resistance to the therapy have been investigated. It has been shown that the administration of interferon has a certain effect on the tumor growth, but it has no effect against tumor recurrence. In patients with a severe clinical course the epithelial atypia of basal layer of papilloma and the increased enzymatic activity have been found.

Genetic characterization of wild type measles virus isolated in Croatia during the 2003-2004 outbreak.

Viral epidemiology is determined by the movement of infected people within and between geographical areas. The genetic characterization of wild-type isolates combined with standard epidemiological methods may enable the identification of the source and transmission pathways and permit differentiation between indigenous and imported viruses. We investigated the genetic characteristics of the wild-type measles virus isolated in Croatia during a 2003-2004 outbreak. The results of this study indicate the presence of the D4 measles virus genotype in Europe. The isolated virus is closely related to virus isolates from the India-like subgroup of the D4 measles virus genotype. The virus responsible for this outbreak differs in the hemagglutinin gene sequence from other virus strains belonging to the D4 genotype. The hemagglutinin gene sequence also differs when compared to viruses from other genotypes that are known to circulate in Europe and from vaccine strains.

In vitro inhibition of macrophage spreading by antigens and lymphokines: correlation with the footpad test and kinetics of inhibition.

As inhibition of spreading of mouse peritoneal macrophages is the basis for an in vitro test of cellular immunity, this test was used to investigate the correlation between in vitro and in vivo results and the kinetics of inhibition of spreading. BALB/c and NIH strain mice were immunized with human or bovine serum albumin or bovine gamma globulin. They displayed a positive footpad test when challenged with the specific antigen (21 days later). Peritoneal cells (PC) of mice with a strong footpad reaction were used for the spreading inhibition test. Intensity of spreading inhibition correlated well (r = -0.93) with that of the footpad reaction. Spreading inhibition had a cyclic pattern. A similar pattern was observed after incubation of PC with preformed lymphokines. The cyclic pattern was probably caused by a repeated action of lymphokines on spread macrophages, resulting in reversible and re-inducible inhibition.

Sendai virus induces various cytokines in human peripheral blood leukocytes: different susceptibility of cytokine molecules to low pH.

Virus infection of cell cultures induces the synthesis of various cytokines which can either inhibit or stimulate virus replication. The Sendai virus induces large quantities of biologically active interferon (IFN-alphan3) in human peripheral blood leukocytes (hPBL) in vitro, as well as many other cytokines. The supernatants of Sendai virus-infected hPBL contained biologically active IFN-alphan3, significant amounts of immunogenic IFN-gamma, monokines (IL-1alpha, IL-beta, TNF-alpha), lymphokines (IL-6, TNF-beta), growth factor (PDGF-AB) and small concentrations of IL-2 and GM-CSF. The analysis of the influence of the Sendai virus inactivation by lowering pH 2.0 on the cytokine concentrations showed that IL-1alpha, TNF-alpha, TNF-beta and IFN-gamma are susceptible to acid conditions, while IFN-alphan3, IL-1beta, IL-6 and IL-2 concentrations remained unchanged.

Immunogenic PDGF-AB dimers in crude leukocyte interferon batches.

The infection of human peripheral blood leukocytes (hPBL) with Sendai virus in vitro, induces the synthesis of human leukocyte interferons (IFN-alpha n3) as well as other cytokines. The authors discovered that supernatants of virus-infected hPBL also contained significant amounts of platelet-derived growth factor AB (PDGF-AB). The concentrations of immunogenic PDGF-AB in the interferon batches were determined by quantitative enzyme-linked immunosorbent assay (ELISA) before and after the inactivation of Sendai virus by acidification (pH 2) for 5 days. Immunogenic PDGF-AB molecules were detected in all interferon batches before and after acidification. The virus inactivation process caused a significant change in the content of immunogenic PDGF-AB in the interferon samples. Inactivation of the Sendai virus by acidification favoured the appearance of PDGF-AB dimers in the majority of samples.

Detection of cellular immunity to tumor-associated antigen(s) in mice by the macrophage spreading inhibition test.

Soluble preparations of antigens from a methylcholanthrene-induced fibrosarcoma of C57BI mice were prepared by homogenization of tumor tissue and high-speed centrifugation of the homogenate. These preparations were able to sensitize syngeneic mice to tumor-associated antigens (TAA) of the fibrosarcoma, and to provoke a delayed hypersensitivity reaction when injected into the footpad of sensitized mice. Furthermore, the same soluble preparations could inhibit in vitro the spreading of peritoneal macrophages from mice sensitized to TAA. A similar inhibition of macrophage spreading was obtained when peritoneal cells from C57BI mice, bearing transplants of the fibrosarcoma, were incubated with the preparations. We conclude that the macrophage spreading inhibition test, like other in vitro assays, can detect cell-mediated immunity to tumor-associated antigens.

Negative effect of uraemia and cuprophane haemodialysis on natural killer cells.

The evidence indicating the important role of natural killer (NK) cells in immune surveillance against tumours and certain infections is accumulating. Uraemic and dialysed patients are known to be at greater risk of infections and malignant diseases. NK cells were analysed in patients with advanced uraemia, and in patients treated with different dialysis techniques. Number of NK cells was morphologically identified as large granular lymphocytes in blood smears. NK activity was determined as mononuclear cell cytotoxicity against K562 cells. In a group of uraemic patients, large granular lymphocyte number was reduced to 39%, and NK activity to 41%-52% of control values. Large granular lymphocyte number and NK activity in patients haemodialysed on cuprophane membranes was significantly reduced, compared to corresponding values in controls and uraemic patients, declining to 17% and 8%-16% of respective control values. In a group of patients treated by CAPD, and in a group haemodialysed on polyacrylonitrile membranes, NK activity was close to values in the uraemic group, but significantly greater than those of cuprophane-haemodialysed patients. Haemodialysis on cuprophane membranes has an additional negative effect on NK cells, which are already seriously depressed by the uraemic state.

Polymorphonuclear leucocyte and natural killer cell functions in mature and premature newborns.

In this study the phagocytic and natural killer cell (NK) functions in 17 premature and 30 mature newborns are compared. The ability of polymorphonuclear phagocytes (PMNs) to ingest, digest and lyse (antibody-dependent cell-mediated cytotoxicity, ADCC) opsonized sheep red blood cells and NK activity were tested. Examinations were performed in cord and venous blood within 6 h or 3-4 days after delivery. Results of examinations were compared with normal values for the group of healthy 4- to 15-year-old children. To assess the influence of the newborn's maturity and age on the tested PMNs and NK functions, the following comparisons were made. (1) Cord vs. peripheral venous blood: only ADCC was higher in peripheral than in cord blood. (2) Mature newborn cells obtained either 6 h or 3-4 days after delivery: ingestion and ADCC were lower and NK activity was higher 3-4 days after delivery. (3) Premature vs. mature newborn cells tested 3-4 days after delivery: ingestion and ADCC were higher while NK activity was lower in premature newborns. (4) Premature newborns tested at 3-4 days vs. mature newborns tested within 6 h after birth: ingestion was lower in the prematures while digestion, ADCC and NK activity were similar. (5) Cells from all newborns tested vs. those of healthy older children: results depend on the interval after birth when newborns were tested. Thus, within the first 6 h after delivery, mature newborns had higher ingestion and ADCC capacity but lower digestion and NK activity. Later, 3-4 days after birth, ingestion, ADCC and NK activity were lower in mature newborns. In the prematures at that interval NK activity was lower. (6) There was a positive correlation between gestational age and NK activity of newborns.

In vitro detection of cellular immunity to melanoma antigens in man by the monocyte spreading inhibition test.

In vitro inhibition of monocyte spreading (a correlate of cellular immunity) was used to detect cell-mediated immune reactions of melanoma patients to specific melanoma antigens. Two soluble preparations of human melanoma antigens (MA-1 and MA-2) and one of a breast carcinoma (BCA) were prepared. The preparations were incubated in vitro with mononuclear cells isolated from the blood of 24 patients with melanoma, six patients with malignancies other than melanoma and 14 healthy donors. Spreading of monocytes from healthy donors was not inhibited by either MA-1 and MA-2 or BCA. MA-1 and MA-2 significantly inhibited the spreading of monocytes from patients with melanoma, while monocytes from patients with other malignancies were not affected. Spreading of monocytes from patients with melanoma was inhibited by the preparation of BCA. We conclude that inhibition of monocyte spreading can detect, in vitro, a cellular immune reaction to specific melanoma antigens in patients with melanoma.