Allergoid-specific T-cell reaction as a measure of the immunological response to specific immunotherapy (SIT) with a Th1-adjuvanted allergy vaccine.

BACKGROUND: Specific immunotherapy (SIT) is believed to modulate CD4+ T-helper cells. In order to improve safety, SIT vaccines are often formulated with allergoids (chemically modified allergens). Interaction between T-cells and allergoids is necessary to influence cellular cytokine expression. There have been few reports on identification the early cellular effects of SIT. METHOD: Patients allergic to grass and/or mugwort pollen (n= 21) were treated with a 4-shot allergy vaccine (Pollinex Quattro) containing appropriate allergoids (grass/rye and/or mugwort) adsorbed to L-tyrosine plus a Th1 adjuvant, monophosphoryl lipid A (MPL). Fourteen grass-allergic patients served as untreated controls. Using the peripheral blood mononuclear cells of these patients, an optimized lymphocyte transformation test (LTT) was employed to monitor the in vitro proliferative response of T-cells to an allergoid challenge (solubilised Pollinex Quattro) before the first and last injection and then 2 and 20 weeks after the final injection. Control challenges utilised preparations of a similar pollen vaccine without the adjuvant MPL and a tree pollen vaccine with and without MPL. RESULTS: The LTT showed increased LTT stimulation indices (SI) in 17/20 SIT patients when the solublised vaccine preparation was used as a challenge before the last injection and 2 weeks after, in comparison to pre-treatment levels. Twenty weeks after therapy, the SI decreased to baseline level. A vaccine challenge without MPL gave lower SI levels. A challenge of a clinically inappropriate tree allergoid vaccine gave no response, and a nontreated group also showed no response. CONCLUSION: Following a short-course SIT adjuvated with MPL, challenges of allergoids were shown to activate allergen-specific T cells in vitro. There was an additional stimulating effect when the challenge was in combination with MPL. There were no non-specific effects of MPL, shown by the tree allergoid/MPL control. The timing of the response was closely correlated to the treatment course; reactivity fell two weeks after the final injection and 20 weeks later it was at baseline level. Thus an immunological response to SIT was detected after very few injections. This methodology could provide a basis for monitoring the immediate progress of allergy vaccinations.

Improving the in vitro antigen specific T cell proliferation assay: the use of interferon-alpha to elicit antigen specific stimulation and decrease bystander proliferation.

The measurement of the proliferative response of primed T cells to an antigenic stimulus (lymphocyte transformation assay: LTT) is commonly used for determining T cell immune responsiveness. However, the ratio between the spontaneous and the antigen-triggered response (stimulation index) is frequently quite low (<3-5) making the interpretation difficult. We modified the assay by the addition of interferon-alpha and the use of fresh autologous serum instead of human AB pool serum. These measures significantly enhanced the stimulation index following stimulation with tetanus toxoid, Candida albicans and tick-borne encephalitis (TBE) viral antigen in studies of sensitized patients. There was no concomitant increase in false positive results. Kinetic studies showed a reduced nonspecific background proliferation of non-stimulated cultures particularly between days 4 and 6 of culture. Furthermore, the positive effect of interferon-alpha were confirmed in studies of patients with contact allergy to nickel and gold. We conclude that this modified form of proliferation assay significantly increases the signal to noise ratio which can be attained. This may be of particular value when looking at T cell responses in immunocompromised patients or in diagnostic attempts to detect very low frequencies of antigen-specific T cells.

A case of severe chronic active infection with Epstein-Barr virus: immunologic deficiencies associated with a lytic virus strain.

Infectious mononucleosis (IM) is a self-limiting, lymphoproliferative disease induced by primary infection with the Epstein-Barr virus (EBV). Infection with EBV leads in general to lifelong asymptomatic persistence of the virus. We report the case of a woman who acquired IM at the age of 15 years and then suffered from recurrent high fever, fatigue, and signs of immunologic disorder for more than 12 years until she died of liver failure. In an attempt to describe and to define the course of chronic active infection with EBV, we performed immunologic and molecular assays that demonstrated lytic replication of EBV in the B and T cells of the peripheral blood. In addition to signs of humoral and cellular immune deficiency, we detected an EBV strain with an impaired capability to immortalize B cells and a tendency to lytic replication, thus contributing to the pathogenesis of this chronic active infection.

Toxoplasma infection and cell free extract of the parasites are able to reverse multidrug resistance of mouse lymphoma and human gastric cancer cells in vitro.

A large number of compounds are known to reduce the ATP-dependent efflux pump activity of multidrug resistant (mdr) tumor cells. Here we report that an infection of cancer cells with T. gondii reduced the multidrug resistance of the tumour cells against cytostatic drugs. Two mouse lymphoma cell lines (Mdr L 5718 and Par 5718) were infected with Toxoplasma gondii in vitro and the reduction of efflux pump activity of the cells was measured. The drug accumulation (Rhodamin-123) was increased in the infected mdr cell lines compared with non- infected mdr-cells, and no effect was shown after infection of the parental cell line. The same effect was also achieved by incubation of Mdr-tumor cells with cell lysate of Toxoplasma gondii. Mdr-1-gene expression was reduced in the infected cell lines 48 hours after infection. Co-cultivation of Toxoplasma gondii with mdr cell lines separated by a microfilter from tumor cells was performed, but this cocultivation did not change the mdr efflux activity. The effect of Toxoplasma gondii infection on the efflux pump activity and mdr-1 gene expression was also examined in the human gastric cancer cells. A sensitization of resistant gastric cancer cells was also achieved by parasite infection. This phenomenon is an evidence that a reduction of resistance in tumor cells can be achieved by a natural parasite infection. It is as yet unclear whether an active infection or another substance of T. gondii is responsible for this phenomenon.

Interferon gamma-1b in the treatment of compensatory anti-inflammatory response syndrome. A new approach: proof of principle.

BACKGROUND: Immunoparalysis is defined as a decrease in the level of HLA-DR expression on monocytes during the course of sepsis. OBJECTIVE: To evaluate whether interferon gamma-1b has an immunoregulatory effect in patients with immunoparalysis during the compensatory anti-inflammatory response syndrome. METHODS: Of the patients admitted consecutively to the intensive care unit for the management of sepsis, 10 received interferon gamma-1b, 100 micrograms per 0.5 mL, after confirmation of HLA-DR expression of less than 30% on 2 consecutive days. The therapy was continued until HLA-DR expression remained more than 50% for 3 days. RESULTS: Interferon gamma-1b therapy resulted in the recovery of diminished levels of HLA-DR expression on monocytes. Of the 10 patients, 8 responded to treatment within 1 day. On the first day of interferon gamma-1b therapy, HLA-DR expression increased from mean (+/- SEM) pretreatment levels of 27% +/- 6% to 62% +/- 8% (P < .01) and remained high during the 28-day study period in 8 patients. The therapy was given to 2 patients a second time when HLA-DR expression on monocytes was less than 30%. The recovery of monocytic HLA-DR expression levels after administration of interferon gamma-1b was associated with restitution of monocytic function, reflected by a significant increase of plasma interleukin-6 (P < .05) and tumor necrosis factor alpha (P < .05) levels in 9 patients. CONCLUSIONS: This study shows that HLA-DR expression is a good marker of compensatory anti-inflammatory response syndrome. It also shows that interferon gamma-1b not only restored the levels of HLA-DR expression but also reestablished the ability of monocytes to secrete the cytokines interleukin-6 and tumor necrosis factor alpha.

Generation and characterization of a human monoclonal IgM antibody that recognizes a conserved epitope shared by lipopolysaccharides of different gram-negative bacteria.

A hybridoma cell line secreting a human monoclonal antibody (humab) directed to an epitope in the lipid A region of lipopolysaccharides of Gram-negative bacteria was isolated. Peripheral blood lymphocytes (PBL) obtained from a healthy volunteer were immortalized by Epstein-Barr virus (EBV) transformation. Lymphoblastoid cell lines (LCL) secreting antibodies to the lipopolysaccharides of Gram-negative bacteria were determined by an enzyme-linked immunosorbent assay (ELISA) and subsequently fused with the human-mouse heteromyeloma cell line CB-F7 by polyethylenglycol (PEG)-mediated fusion. A hybridoma line producing a humab (LPD5H4), of the IgM/lambda isotype, which strongly reacted with the lipid A portion of Salmonella and E. coli spp. in ELISA, was established. The antibody was purified by hydrophobic interaction chromatography and gel filtration. Immunoblotting experiments showed a strong reactivity of the humab LPD5H4 with the lower molecular species of different rough and smooth lipopolysaccharide (LPS) types of the bacteria species Salmonella, E. coli, Klebsiella, and Neisseria meningitidis, whereas those of Pseudomonas spp. were negative. Binding of humab LPD5H4 to solid phase bound lipid A and different rough mutants of LPS could be inhibited by the corresponding antigens in solution. Competition assays with a murine monoclonal antibody to lipid A and with polymyxin B indicate that humab LPD5H4 recognizes its epitope in this extremely conserved part of the LPS molecule. In vitro tests demonstrated that the MAb is able to partially inhibit the LPS-induced release of TNF-alpha using isolated peripheral blood mononuclear cells (PBMC).

Immunodepression following neurosurgical procedures.

OBJECTIVE: To determine the influence of a selective, sterile central nervous system surgery on immune reactivity, particularly whether a decrease of monocytic human leukocyte antigen-DR expression, indicating immunodepression, occurs after neurosurgery and if this measurement is useful for identification of patients with a high risk of infection. DESIGN: Prospective study. SETTING: Department of neurosurgery and intensive care unit in a university hospital. PATIENTS AND INTERVENTIONS: Blood samples were obtained from 46 patients at least once during the first 3 days after undergoing sterile central nervous system surgery. Fourteen of these patients developed infectious complications as defined by clinical and microbiological criteria. In ten of 46 patients, paired samples of blood and cerebrospinal fluid were collected from a ventricle drain at the following times: 1 day before surgery; several times on the day of surgery; and every day after surgery for at least 6 days. MEASUREMENTS AND MAIN RESULTS: Monocytic human leukocyte antigen-DR expression, as measured by flow cytometry on days 1 through 3 after surgery in 46 patients, was lower in 14 patients who developed infection after neurosurgery (p < .0001). In all ten closely monitored patients, monocytic human leukocyte antigen-DR expression decreased temporarily after surgery. Of these patients, only one patient showed a persistent and considerably decreased monocytic human leukocyte antigen-DR expression. This patient was the only patient in this subgroup who developed sepsis syndrome. In order to assess whether the monocytic human leukocyte antigen-DR decrease was associated with a preceding inflammatory response, local and systemic concentrations of interleukin (IL)-1 beta, IL-6, IL-8, tumor necrosis factor-alpha, and interferon-gamma were measured in this subgroup. These cytokines were not detectable in plasma during the first days after surgery. In contrast, considerable increases of IL-6 and IL-8 concentrations were detectable in cerebrospinal fluid within hours after surgery. CONCLUSIONS: A decrease of monocytic human leukocyte antigen-DR expression occurs after neurosurgery and is associated with a preceding, strong, intracranial (but not systemic) inflammatory response. A very low monocytic human leukocyte antigen-DR expression (< 30% positive monocytes) suggests the possibility of infection. Measurement of monocytic human leukocyte antigen-DR expression could help to detect patients with a high risk of infection after neurosurgery. Our results suggest that even sterile central nervous system surgery may contribute to general immunodepression. The local intracranial inflammatory response may be involved in this process.

Cell cluster formation during up-scaling of a human-mouse heterohybridoma producing a polyspecific human IgM antibody.

Up- and downstream processing of human monoclonal IgM is known to bring about problems with respect to clone stability and quantity of antibodies produced. A human B cell hybridoma producing a natural polyreactive IgM antibody (CB03) was adapted to growth in serum-free medium and scaled-up using a hollow fiber bioreactor system. The process of fermentation has been carried out continuously over a period of 4 months. In comparison to stationary culture conditions in the presence of 10% fetal calf serum, antibody concentrations in hollow fiber bioreactor supernatants were found to be significantly increased. Semicontinuously harvested supernatants contained up to 400 mg/liter immunoreactive IgM antibody. During the last weeks of fermentation, a markedly reduced number of viable cells was observed, whereas antibody production seemed to remain stable. Furthermore, we detected formation of cell clusters in the fermentor system. These clusters carried IgM on the surface and secreted immunoreactive IgM antibodies. Clusters were found to represent fusions of hybridoma cells using electron microscopy. Cluster formation was accompanied by decreased glucose consumption and lactate accumulation and was not seen during growth of other human hybridomas. We discuss these results in the content of the polyreactive binding properties of this particular antibody.

Introduction of the octanucleotide restriction site SwaI into the bicistronic vector pTiSDT for high level synthesis of proteins.

The octanucleotide recognition site for the endonuclease SwaI was introduced into the Escherichia coli bicistronic expression vector pTiSDT by mutating a single position in the coupling SD sequence between a truncated form of the cro-gene and the multicloning site. This mutation does not influence the expression rate. The introduction of this restriction site allows high level production of proteins, that are modified only by an N-terminal methionine incorporated as the start codon.

Monoclonal antibodies to p24-core protein of HIV-1 mediate ADCC and inhibit virus spread in vitro.

BACKGROUND: Certain antigens of the HIV-1, e.g., gp120-envelop proteins, can be expressed on the membrane of HIV-infected cells. Little is known about the membrane expression of other HIV-antigens and their interaction with specific antibodies. OBJECTIVE: To develop murine monoclonal antibodies (mAbs) to the p24-core protein of HIV-1 and to characterise their binding sites and biological activities on HIV-infected T cells. METHODS: Monoclonal antibodies were developed from mice hyperimmunised with a recombinant p24-core protein from HIV-1. Two mAbs were epitope-mapped on overlapping peptides and characterised for their reactivity with non-fixed HIV-infected T cells by immunofluorescence staining and flow cytometric analysis. Their biological activities were studied for antibody-dependent cellular cytotoxicity (ADCC) and suppression of viral spread in vitro. RESULTS: The epitopes of two selected mAbs were located on the amino terminal region of p24 in the regions 147-152 aa and 178-187 aa, respectively. The antibodies were able to react with living HIV-1 infected cells. The expression of the antigens was time-dependent after the infection of certain cell lines by HIV-1. The mAbs mediated a strong HIV-1-specific ADCC and were able to delay the spread of HIV-1 for about 6 days in cell cultures. CONCLUSIONS: Certain epitopes of the p24-core protein of HIV-1 can be expressed on living, HIV-infected T cells and are recognised by specific antibodies. Such antibodies can destroy infected cells by ADCC or delay the virus spread, and therefore, should be considered in immunisation strategies against HIV.

Identification and characterization of a TNF alpha antagonist derived from a monoclonal antibody.

Peptides derived from the CDRs of the anti-TNF alpha monoclonal antibody Di62 were tested for inhibition of binding of Di62 to TNF alpha as well as of TNF alpha to its 55 and 75 kDa receptor. A peptide derived from the CDR1 of the light chain was shown to specifically inhibit Di62 binding to TNF alpha with markedly higher activity (Ki = 4 microM) than all other CDR-derived peptides. This peptide also significantly inhibited binding of TNF alpha to its 55 and 75 kDa receptor and protected L929 cells from the cytotoxic effect of TNF alpha (IC50 = 6 microM). The C-terminal region of this peptide, which is homologous to the 55 and 75 kDa TNF receptor, was found to be essential for activity.

Cytomegalovirus infection in transplant recipients. The role of tumor necrosis factor.

Human cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in transplant recipients. CMV infection commonly results from the reactivation of a latent infection. Using a set of monoclonal anti-CMV antibodies, we found CMV antigen expression in peripheral blood mononuclear cells (PBMNC), particularly in monocytes, in 312 of 816 samples from 190 allograft recipients. The detection of CMV-IE antigens and CMV-IE DNA in PBMNC indicates that positive cells may represent truly infected cells. The relation between increased cytokine plasma levels (particularly following treatment by pan-T cell antibodies) and the appearance of CMV antigens in PBMNC suggests that cytokines may play an important role in the reversal of CMV latency. This hypothesis is supported by our finding that tumor necrosis factor-alpha (TNF) is able to stimulate the activity of the CMV-IE enhancer/promoter region in the human monocytic cell line, HL-60. The interleukins 1, 2, 3, 4, 6, 8 and 10; transforming growth factor-beta; interferongamma; and granulocyte/macrophage colony-stimulating factor did not show any enhancing effect on the CMV promoter activity. Thus, TNF-alpha seems to play a key role in regulating the balance between latency and reactivation of CMV infection. Inhibition of TNF-alpha release or action may be an alternative strategy for preventing CMV-associated morbidity in allograft recipients.

Characterization of neutralizing monoclonal antibodies directed against Staphylococcus aureus alpha-toxin.

A panel of neutralizing murine monoclonal antibodies (MAbs) against Staphylococcus aureus alpha-toxin has been established, using formaline-inactivated alpha-toxin as an immunogen. Five independent groups of neutralizing epitopes have been identified representing five functionally important structures in the toxin molecule. Because none of the antibodies binds to overlapping decapeptides representing the toxin sequence or to bromocyanogen cleavage products of alpha-toxin, they may all bind to conformational epitopes. Nevertheless, they all bind to monomeric alpha-toxin in a Western blot. Three of the antibodies bind to the toxin monomer in an enzyme-linked immunosorbent assay (ELISA) in the presence, but not in the absence, of detergent. These epitopes are not accessible in hexameric toxin; two of them may represent the contact sites of the toxin monomers upon hexamerization and one is related to a structurally important glycine-rich central hinge region. Two different antibodies bind to monomeric toxin in an ELISA in the presence and absence of detergent and their epitopes are present more than once on oligomeric toxin; they bind strongly to hexameric toxin in a Western blot. The binding properties of the antibodies to alpha-toxin in different assay systems are summarized in an epitope model, which describes the presence of neutralizing domains in the different conformational steps required for pore formation.

Characterization of a B-CLL derived IgM-lambda antibody expressing typical features of a NPAB.

A human IgM-lambda hybridoma (CB-HB) was established from chronic lymphocytic leukaemia (CLL) B-cells. Immunochemical and molecular characterization of the monoclonal antibody (mab) produced by the CB-HB cells offered typical features of natural polyreactive antibodies (NPAPs) found in fetal and healthy adult organisms. In particular, the CB-HB mab reacted with different self and foreign (viral and bacterial) antigens when tested in three independent systems (solid- and fluid-phase ELISA, Western blot) showing binding constants in a range from 1.9 x 10(-7) to 7.5 x 10(-8) mol/l to four antigens chosen. In addition, the CB-HB mab binding could be inhibited by a rabbit polyclonal antiserum specific for a common idiotype (Id 102) on human polyreactive (auto)antibodies. The variable region of the CB-HB mab was found to be encoded by unmutated copies of germline genes. Interestingly, the VH-DP10 (51p1) segment, encoding for the autoantibody-associated G6-cross reactive idiotype frequently expressed on both fetal and malignant B-cells, was found to be used with a V segment (VLO11). Collectively these data imply that cells belonging to the natural polyreactive B-cell repertoire undergo malignant transformation. A stimulation by autoantigens or common foreign antigens may be involved.

Interleukin-6 and interleukin-8 concentrations as predictors of outcome in ventricular assist device patients before heart transplantation.

OBJECTIVE: To determine whether the serum concentrations of some circulating cytokines (as highly sensitive markers of inflammation) are of value in predicting the outcome of patients with cardiogenic shock and end-stage heart disease, who undergo ventricular assist device implantation until heart transplantation. DESIGN: Cohort study. SETTING: University teaching hospitals. PATIENTS: Twenty patients with cardiogenic shock or end-stage heart disease were consecutively selected for this study, if assist device implantation was performed as a bridge to heart transplantation. MEASUREMENTS AND MAIN RESULTS: The circulating concentrations of the cytokines interleukin (IL)-1 beta, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha were monitored from the beginning to the end of assist device support two to three times a week, using commercial enzyme-linked immunosorbent assays (ELISA). In all patients, circulating IL-6 and IL-8 values were increased shortly after assist device implantation. In patients with uncomplicated courses, IL-6 and IL-8 concentrations decreased after an initial increase and were low at the time of transplantation, whereas serum cytokine concentrations increased and remained increased in the nonsurvivors (survivors vs. nonsurvivors, p < .001). Circulating IL-1 beta and TNF-alpha concentrations were rarely detectable. CONCLUSIONS: Monitoring of IL-6 and IL-8 values during ventricular assist device support provides a means of early identification of high-risk patients that may allow optimization of antimicrobial therapy and selection of the appropriate time for transplantation.

Binding of natural human IgM auto-antibodies to human tumor cell lines and stimulated normal T lymphocytes.

In a recent publication we described the binding of natural IgM antibodies derived from the human fetal B cell repertoire to the cell surface of some human tumor cells including colon carcinoma, small-cell lung cancer and B lymphoma lines [1]. Further analyses showed that a similar molecule was bound by the respective monoclonal human antibodies on the cell surface of polyclonally stimulated human CD3+ T cells, but is absent from unstimulated MNC. Both CD4+ and CD8+ stimulated cells were recognized. The molecule was found to be expressed together with lymphocyte activation markers (4F2, CD72, CD25). The membrane antigen expressed on both the activated T lymphocytes and tumor cells was characterized in a 2-D electrophoresis system: molecular weight 55-60 kDa, pI-approximately 6.0. Whereas the proliferation capacity of tumor cells was detected to be decreased significantly in the presence of the binding antibodies, no influence on [3H]thymidine uptake into stimulated T cells was found, suggesting different functional consequences of binding the respective antigen on malignant and normal cells. An interesting finding is the enhanced expression of major histocompatibility complex class I molecules on tumor cells incubated with human natural antibodies.

Monitoring of the cellular immune system in patients with biventricular assist devices awaiting cardiac transplantation.

Lack of objective parameters to predict the clinical course and outcome are a major problem in managing the patients selected for BVAD-support as a bridge to heart transplantation. This study was intended to assess whether cellular immune parameters have a predictive value for the clinical result of VAD-support. Various cellular immune markers were monitored by multiparameter cytofluorometry in 30 patients who received a VAD system (Berlin Heart). We did not find significant differences in preoperative values of immune parameters between groups of survivors (n = 14) and non-survivors (n = 16). All 9 patients who died of septic multiple organ failure (MOF) had shown increased levels of T-cell activation (CD 71, CD 25, HLA-DR) as well as leukocytosis and 7 patients who died of noninfectious complications (mostly hemorrhage or cerebral complications) had exhibited T-lymphopenia. Seven of 9 patients who died of septic MOF had extremely decreased levels of HLA-DR+ monocytes (< 30%) while all 14 survivors and all 7 patients who died of noninfectious complications showed almost normal monocytic HLA-DR antigen expression, antigen-presenting capacity and cytokine secretion. These observations point to the reduced antimicrobial immunity ("immunoparalysis") in the non-survivors and may explain the fatal course of infection in these individuals. The in vitro results of restitution experiments call for new therapeutic strategies to improve the survival of VAD-patients.

VH/VL gene expression in polyreactive-antibody-producing human hybridomas from the fetal B cell repertoire.

Among a panel of nearly 3,000 IgM-producing hybridomas obtained from 22 independent fusions of human fetal lymphocytes (liver/spleen; 15th-36th gestational week) a high number (5-10%) produced autoantibodies, independently of the gestational age. A significant portion of these autoantibodies was found to be polyreactive, i.e. capable of binding to more than two antigens, when tested against a set of five antigens of the internal (ssDNA, thrombocytes, keratin) and external (lipid A, tetanus toxoid) environment. Analyzing the IgVH genes utilized in eight polyreactive and two putatively nonpolyreactive hybridomas, members of the VHI, III, IV and VI families were found once, seven times, once and once, respectively, mostly with germline identity. All but one of the utilized gene elements could be related to the biased VH gene repertoire said to be expressed during the early ontogeny of the human immune system. We also noted a bias for the utilization of DN1 (3/10), DHQ52 (3/10), JH2 (4/10) and JH6 (4/10) elements, whereas all heavy-chain CDR3 regions manifest a diversity by addition of N nucleotides and/or exonuclease activity on coding segments. In addition, VL segments which belong to different subgroups of both isotypes were found to be used. The molecular basis of polyreactive immunoglobulin specificities in human fetuses is discussed.