Immunotherapy using lipopolysaccharide-stimulated bone marrow-derived dendritic cells to treat experimental autoimmune encephalomyelitis.

Lipopolysaccharide (LPS) produced by Gram-negative bacteria induces tolerance and suppresses inflammatory responses in vivo; however, the mechanisms are poorly understood. In this study we show that LPS induces apoptosis of bone marrow-derived dendritic cells (DCs) and modulates phenotypes of DCs. LPS treatment up-regulates expression of tolerance-associated molecules such as CD205 and galectin-1, but down-regulates expression of Gr-1 and B220 on CD11c(+) DCs. Moreover, LPS treatment regulates the numbers of CD11c(+) CD8(+) , CD11c(+) CD11b(low) and CD11c(+) CD11b(hi) DCs, which perform different immune functions in vivo. Our data also demonstrated that intravenous transfer of LPS-treated DCs blocks experimental autoimmune encephalomyelitis (EAE) development and down-regulates expression of retinoic acid-related orphan receptor gamma t (ROR-γt), interleukin (IL)-17A, IL-17F, IL-21, IL-22 and interferon (IFN)-γ in myelin oligodendrocyte glycoprotein (MOG)-primed CD4(+) T cells in the peripheral environment. These results suggest that LPS-induced apoptotic DCs may lead to generation of tolerogenic DCs and suppress the activity of MOG-stimulated effector CD4(+) T cells, thus inhibiting the development of EAE in vivo. Our results imply a potential mechanism of LPS-induced tolerance mediated by DCs and the possible use of LPS-induced apoptotic DCs to treat autoimmune diseases such as multiple sclerosis.

Tolerogenic dendritic cells produced by lentiviral-mediated CD40- and interleukin-23p19-specific shRNA can ameliorate experimental autoimmune encephalomyelitis by suppressing T helper type 17 cells.

Down-regulation of soluble or membrane-bound co-stimulatory molecules by RNAi in dendritic cells can prevent the activation of immune responses. Therefore, this study was designed to evaluate the therapeutic efficacy of bone marrow-derived DCs (BMDCs) transduced with lentiviral vectors to permanently expressed shRNA specific for CD40 (CD40LV-DCs) and/or p19 subunit of interleukin (IL)-23 (p19LV-DCs) mRNAs in experimental autoimmune encephalomyelitis (EAE). In-vitro studies showed that double-transduced BMDCs (CD40(+) p19LV-DCs) resemble tolerogenic DCs due to profound down-regulation of CD40, lower expression of proinflammatory cytokines (IL-6 and IL-12), increased IL-10 production and stronger stimulation of myelin oligodendrocyte glycoprotein (MOG)35-55 -specific T cells for production of IL-10 compared with CD40LV-DCs, p19LV-DCs and BMDCs transduced with control lentiviral vector (CoLV-DCs). Moreover, injection of transduced CD40(+) p19LV- BMDCs in EAE mice resulted in more reduction in clinical score, significant reduction in IL-17 or increased production of IL-10 by mononuclear cells derived from the lymph nodes or spinal cord compared with CoLV-DCs-treated EAE mice. In conclusion, simultaneous knock-down of CD40 and IL-23 production by BMDCs may represent a promising therapeutic tool for the treatment of IL-17-dependent autoimmune diseases, including multiple sclerosis.

Immunomodulation using the recombinant monoclonal human B7-DC cross-linking antibody rHIgM12.

A patient with Waldenstrom's macroglobulinaemia expresses a high titre IgM antibody in serum that binds both mouse and human dendritic cells (DC) in a B7-DC (PD-L2)-dependent manner. We have reported previously that purified antibody from patient serum activates immature and mature DC in vitro, enhancing the ability of these professional antigen-presenting cells to activate naive T cells, take up antigen, resist a cytokine-depleted environment and secrete immunomodulatory cytokines, such as interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha. Systemic treatment of experimental animals with this antibody induces potent anti-melanoma immunity and modulates protectively the recall response against antigen challenge through the airway in an experimental model of inflammatory airway disease. Here we describe a monoclonal IgM antibody derived from this serum immunoglobulin that recapitulates each of these earlier observations, providing direct evidence that M protein from the Waldenstrom's patient mediates these potent immunomodulatory effects. Furthermore, cell lines expressing this recombinant form of the human antibody provide the basis for developing this reagent for clinical application.

Immunoglobulin-mediated CNS repair.

Our view of the immune system continues to evolve from a system dedicated primarily to defense against pathogens to a system that monitors the integrity of the organism and aids in repair following damage. Repair following injury to the central nervous system (CNS) is facilitated by both cellular and humoral components of the immune system. Transfer of macrophages or T cells activated against CNS antigens promote axon regrowth and protect axons from further damage. Animals immunized with spinal cord antigens and subsequently challenged with demyelination or transection of the spinal cord demonstrate better repair than animals without prior immunization. In both experimental systems, antibodies are the biologically active immune component. Human mAbs reactive to oligodendrocytes that arise in the absence of neurologic injury promote remyelination. These data support the hypothesis that B-cell clones producing mAbs reactive to CNS epitopes are a normal part of the human antibody repertoire. They challenge the assertion that an immune response to CNS antigens is pathogenic. Treatment with CNS-reactive human mAbs following CNS disease may facilitate CNS regeneration.

MSP, a trypsin-like serine protease, is abundantly expressed in the human nervous system.

The goal of the present investigation was to determine the regional and cellular specific expression patterns of the newly identified serine protease, myelencephalon-specific protease (MSP), in the adult human brain (Scarisbrick et al. [1997b] J. Neurosci. 17:8156-8168). To assess the potential scope of MSP activity, Northern blot techniques were used to determine the relative abundance of MSP mRNA in 16 different adult human brain regions, and in the brain and peripheral tissues of the midgestation human fetus. The regional and temporal specific expression patterns of MSP mRNA were directly compared with those of tissue plasminogen activator (tPA), a serine protease strongly implicated in the development, ongoing plasticity, and response of the nervous system to injury and disease. mRNA encoding each protease was distributed widely throughout the normal adult human central nervous system (CNS), but the expression of each was only partially overlapping. Additionally, compared with tPA, MSP exhibited a more restricted distribution and delayed developmental onset. By immunohistochemical localization, MSP was present at moderate to high levels in neurons and oligodendroglia of the adult human brain, at a level closely resembling the relative abundance indicated by Northern blot. MSP was most abundantly expressed in the spinal cord, hippocampus, substantia nigra, and basal ganglia. The robust expression of MSP in clinically significant regions of the adult human CNS indicates that further study of this protease in terms of both normal brain physiology and neurodegenerative disorders is warranted.

Clonal evolution in Waldenstrom macroglobulinemia highlights functional role of B-cell receptor.

The course of clonal evolution of 2 related clones in the blood of a patient with Waldenstrom macroglobulinemia (WM) indicates the functional importance for the expression of the B-cell receptor for the survival of these malignant cells. Protein and nucleotide sequencing of the paraproteins' variable regions revealed 2 predominant Vlambda and 2 VH sequences, each set comprised in the ratio 1:1.5. The 2 VH sequences and 2 Vlambda sequences shared the same VDJ and VJ junctional sequences, respectively, indicating that 2 malignant clones had evolved from a common ancestor. This is the first report on intraclonal heterogeneity in WM. Comparison of the Vlambda and VH sequences with the closest matching known germline genes showed that they contained approximately 10 somatic mutations each. The distribution and type of mutations demonstrate that mutations have continued to accumulate in the malignant clones and that selection has been operating to preserve immunoglobulin structure.

Human monoclonal antibodies reactive to oligodendrocytes promote remyelination in a model of multiple sclerosis.

Promoting remyelination, a major goal of an effective treatment for demyelinating diseases, has the potential to protect vulnerable axons, increase conduction velocity, and improve neurologic deficits. Strategies to promote remyelination have focused on transplanting oligodendrocytes (OLs) or recruiting endogenous myelinating cells with trophic factors. Ig-based therapies, routinely used to treat a variety of neurological and autoimmune diseases, underlie our approach to enhance remyelination. We isolated two human mAbs directed against OL surface antigens that promoted significant remyelination in a virus-mediated model of multiple sclerosis. Four additional OL-binding human mAbs did not promote remyelination. Both human mAbs were as effective as human i.v. Ig, a treatment shown to have efficacy in multiple sclerosis, and bound to the surface of human OLs suggesting a direct effect of the mAbs on the cells responsible for myelination. Alternatively, targeting human mAbs to areas of central nervous system (CNS) pathology may facilitate the opsonization of myelin debris, allowing repair to proceed. Human mAbs were isolated from the sera of individuals with a form of monoclonal gammopathy. These individuals carry a high level of monoclonal protein in their blood without detriment, lending support to the belief that administration of these mAbs as a therapy would be safe. Our results are (i) consistent with the hypothesis that CNS-reactive mAbs, part of the normal Ig repertoire in humans, may help repair and protect the CNS from pathogenic immune injury, and (ii) further challenge the premise that Abs that bind OLs are necessarily pathogenic.

Human monoclonal IgM DJ binds to ssDNA and human commensal bacteria.

In this study we tried to elucidate further the crossreactivity pattern and binding characteristics of human monoclonal IgM DJ which is an anti-DNA antibody and possesses Y7 natural idiotope. Isolated IgM DJ and its enzymatically obtained fragments Fab' and (Fab')2 were tested for binding to more than 26 antigens and nine bacteria in indirect ELISA. Inhibition of binding studies and examination of the stability of antigen-antibody complexes were also done in ELISA assay. IgM DJ bound to single stranded DNA and human lactic acid bacteria, such as L. acidophyllus, B. bifidum and L. plantarum. This binding was shown to be mediated through IgM DJ Fab' fragment. High avidity and low affinity of interactions was estimated from the binding curves of Fab', (Fab')2 fragments and whole IgM. The common epitopic motif on both antigens were negatively charged phosphodiester moieties. Complexes formed with ssDNA and B. bifidum were resistant to washing with high salt. This suggested that electrostatic attraction was not a strong component of the binding. A novel pattern of natural autoantibody reactivity in a human system related to cross-reactivity with DNA and LAB is described. Possible involvement of LAB in induction of natural anti-DNA antibodies is discussed.

Late apical filling by color Doppler M-mode during dipyridamole stress echocardiography: a useful contribution to wall motion analysis.

It has been shown that regional myocardial ischemia during angioplasty is associated with retarded apical filling. To test the importance of retarded apical filling by color Doppler M-mode to detect ischemia during dipyridamole stress echocardiography, we evaluated 29 patients (12 women, aged 57 to 87 years). High-dose dipyridamole (0.84 mg/kg for 10 minutes) was used. The color M-mode record was used to calculate the duration of abnormal apical flow as measured from the onset of the QRS complex to the disappearance of color signals directed toward the apex. Echocardiographic images were compared at rest and during stress to identify the presence of new or worsening wall motion abnormalities (WMAs). Fourteen patients (group A) were designated as having coronary artery disease on the basis on WMAs during the stress test and abnormal coronary anatomy. Fifteen patients (group B) without WMAs in the presence of normal coronary anatomy were designated as having no coronary artery disease. All but two patients in group A had an abnormal apical filling response to dipyridamole stress (sensitivity 86%). In these patients the marked retardation of apical filling was detected during ischemia (55 +/- 18 msec versus 120 +/- 34 msec) (p <0.01). In group B there were no dynamics in apical filling (specificity 100%). Color M-mode Doppler imaging showed retarded apical filling during dipyridamole-induced myocardial ischemia. This abnormal filling pattern may be a useful adjunct to WMAs during dipyridamole stress echocardiography.

Effect of valency on binding properties of the antihuman IgM monoclonal antibody 202.

A murine monoclonal IgG2a antibody, 202, specific for human IgM, was produced and immunochemically characterized. Binding features of MAb 202, epitope localization, and its accessibility at the quaternary structure of polymeric IgM were investigated. Direct and competitive ELISA with fragments of IgM molecule demonstrated that the epitope recognized by MAb 202 lies on the Fc3 portion of IgM. Sandwich ELISA with MAb 202, which could be used simultaneously to capture and to detect bound IgM, indicated that more than one 202 epitope is present on the IgM molecule. MAb 202 did not precipitate IgM in solution, whereas good precipitation lines were obtained in agarose gel. Binding of MAb 202 to the J chain, C-terminal tailpiece and C mu 2 peptide, which remain attached to the C mu 3 domain of the Fc5 fragment, was excluded by a number of experimental results and structural reasons. Therefore a potential candidate for epitope 202 expression was the C mu 3 domain. MAb 202 did not react with isolated mu chain, which is expected since epitope 202 is of a conformational type. Furthermore, the reaction with monomeric IgM was almost undetectable as was demonstrated by a number of methods (ELISA, immunofluorescence, Western blotting). Since monovalent Fab portions of MAb 202 weakly reacted with polymeric IgM, we concluded that intrinsic affinity of their interaction is low but greatly enhanced by bivalent binding. Antipolymeric IgM binding specificity of MAb 202 was demonstrated only in the case of bivalent binding with a functional affinity constant of Kd = 2.14 x 10(-9) M-1. This implied up to a 10(4) difference between intrinsic and functional affinity, as in the range of concentration used in this study MAb 202 did not react with monomeric IgM.

[Combined myocardial infarct of the left and right ventricle]. / Kombinovani infarkt miokarda leve i desne komore.

Combined myocardial infarction of left and right ventricle (CMILRV) of inferoposterior localization represents simultaneous acute necrosis of both myocardial ventricles and belongs, due to associated and frequent complications, to more severe damages of the myocardium with high mortality. Our group consisted of 100 patients of both sexes, mean age of 67.4 years, of which 50 were with inferoposterior left ventricular infarction and 50 with CMILRV of the same localization. The diagnosis was confirmed clinically, by ECG, by the analysis of enzymes, echocardiographically and scintigraphically using radionuclide ventriculography with 99Tcm PyP. Selective coronarography was performed in all the survived patients, and autopsy of the dead. On the basis of clinical presentation of some forms of dynamic weakness of the heart, in acute stage of CMIRLV, we have established that there are 4 categories of these patients, so that each of these groups represents also a special clinical form, which are: 1. CMIRLV without signs of heart failure, established in 20 (40%) cases, which clinically and prognostically do not differ from inferoposterior left ventricular infarction. 2. CMIRLV with predominant failure of the right ventricle was found in 17 (34%) of the patients with a specific clinical feature and the way of treatment. 3. CMIRLV with marked failure of the left ventricle, found in 8 (16%) of the patients, which required combined treatment measures. 4. CMIRLV with general heart failure, established in 5 (10%) cases, is characterized by cardiogenic shock and falls into the most severe forms of this kind of myocardial infarction. Total mortality in the patients with CMILRV was 10% (5.50), that is 40% (2.5) in both the second and the fourth subgroup, 20% (1/5) in the third group, while in the first one as well as in the control group with inferoposterior infarction there was no mortality. Good knowledge of the above mentioned clinical forms of CMILRV are necessary prerequisite for application of specific and corresponding therapeutic measures and the best prevention of associated complications and high mortality of such patients.

[The obstruction syndrome in patients with bronchogenic carcinoma]. / Opstrukcijski sindrom u bolesnika sa bronhogenim karcinomom.

In the period 1985-1990, 760 patients with bronchogenic carcinoma have been examined and treated at the Clinic of Lung Diseases of the Military Medical Academy. Obstruction syndrome was found in 64% of patients of whom 21% with severe obstruction. Pulmonary insufficiency, most commonly of milder, partial type, was found in 19% of patients. Due to the limited lung function as a consequence of chronic obstructive lung disease (COLD) 45 patients with locally demarcated tumours were not operated, surgical resection was not possible. In the operated patients with obstruction syndrome the risk of postoperative complications was increased. COLD and limited lung function most commonly had not been the obstacle for application of radiotherapy of bronchogenic carcinoma.

Immunochemical characterisation of a murine monoclonal anti-idiotypic antibody.

Y7, a murine monoclonal IgG1 kappa antibody against a human monoclonal IgM lambda DJ molecule, was affinity purified on an IgM lambda immunoaffinity column. As detected by enzyme-linked immunosorbent assay (ELISA) the isolated Y7 monoclonal antibody was shown to be not cross-reactive with human IgG, human secretory IgA, mu chain, lambda + kappa chains and another human monoclonal IgM lambda BR. Binding to the polyclonal human IgM standard in the same assay was about 30 percent. The epitope specificity of affinity purified and biotinylated Y7 MoAb was localized only in the nonreduced pepsin Fab fragments of IgM lambda DJ immunogen. As the immunogen was determined to be a specific antibody to phosphorylcholine, the specificity of Y7 MoAb was further ascertained in its capacity to induce 95% inhibition of immunogen binding for phosphorylcholine.

[Results of combined therapy of small cell bronchogenic carcinoma in a 5-year period (1984-1988)]. / Rezultati kombinovanog lecenja bronhogenog karcinoma malih celija u petogodisnjem periodu (1984-1988).

Within the five-year period (1984-1988) the oat cell bronchogenic carcinoma was diagnosed and treated in 151 patients. Analysed were 74 patients having received complete treatment. Patients with disseminated form received only polychemotherapy in 6 cycles (cyclophosphamide, doxorubicin, vincristine and etoposide) and those with limited disease also received additional, local-regional radiotherapy of 45-50 Gy. In complete respondents preventive brain irradiation was applied. In selected patients with initial forms of the disease surgical resection with additional polychemotherapy were applied. The best survival showed respondents with limited disease and operated patients. Average survival of the whole group was 72 weeks. One year survived 31 out of 74 (41.9%) patients and two years survived 10 out of 74 (13.5%) patients.

[Histopathological evaluation of the endometrium in premenopausal bleeding]. / Histopatoloska evaluacija endometrijuma kod krvavljenja u premenopauzi.

A total of 783 women aged 41-50 were examined in the Out-Patient Clinic in Novi Sad in the period from 1975-1987 due to prolonged uterine bleeding and the explorative curettage was performed in 12 (7%) of them. The most frequent histopathologic results were as follows: hiperplasio glandularis endometrii simplex in 25 (22.3%), hyperplasio glandularis cystica endometrii in 17 (15.7%), hyperplasio adenomatoides endometrii in 12 (10.7%), endometritis chronica in 18 (16%), phasis secretionis endometrii in 7 (6.2%), phasis proliferationis endometrii in 3 (2.6%), endometritis acuta non specifica post graviditates in 5 (4.4%), residua decidualis necrotica cum charione in 8 (7.1%), mola hydatidosa in 1 (0.9%), polypus endometrialis in 3 (2.6%), and adenocarcinoma endometrialis in 3 (2.6%). Uterine bleeding is a very frequent appearance in women's premenopausal period and apart from gynecologic survey it also necessitates the application of diagnostic methods and interventions such as the curretman's histologic investigation intended for the detection of pathologic changes in the endometrium.