Effect of ex vivo cytokine treatment on human cord blood engraftment in NOD-scid mice

Umbilical cord blood transplantation is considered an alternative to traditional bone marrow transplantation for patients who do not have matched sibling donors. In this study, we examined the effects of ex vivo treatment of human cord blood cells with cytokine mixtures and assessed the ability of treated cells to engraft in NOD-scid mice. We incubated the cord blood with a four-factor cytokine mixture of interleukin (IL)-3, IL-6, IL-11 and stem cell factor, or with a two-factor cytokine mixture of thrombopoietin and flt-3. Incubation of cord blood for 48 h with either cytokine mixture did not affect progenitor cell number or proliferative potential as measured by the high proliferative potential (HPP) assay. Cytokine-treated cord blood injected into irradiated NOD-scid mice resulted in multilineage human engraftment. Overall, incubation with cytokines resulted in variable levels of engraftment with different cord blood samples. Incubation of cord blood with the four-factor cytokine mixture resulted in increased survival of irradiated NOD-scid recipients. These results demonstrate that short-term ex vivo treatment of human progenitor cells gives variable results on in vivo multipotential capabilities.     

Homing of human autoreactive T cells into pancreatic tissue of NOD-scid mice

Aims/hypothesis An important prerequisite for the initiation of pancreatic islet inflammation is the recruitment of pathogenic T cells. We investigated the in vivo migration patterns of human islet-reactive T cell clones after transfer into compromised hosts.Methods NOD-scid mice were injected with a mixture of human autoreactive T cells and antigen-presenting cells. Survival and migration of T cells was analysed by fluorescence-activated cell sorter and immunohistochemical analysis of various tissues.Results Autoreactive T cells and antigen-presenting cells survived at least 14 days in vivo and accumulated in spleen, pancreatic tissue and pancreas draining lymph nodes, but not elsewhere, as early as 4 days after transfer. This homing was dependent on co-injection of human antigen-presenting cells loaded with autoantigen. Finally, we found that this process is enhanced by streptozotocin treatment. Streptozotocin treatment did not affect the constitutive homing to pancreas draining lymph nodes. Histological analysis of pancreatic tissue sections showed some autoreactive T cells around the islets of Langerhans, comparable to early peri-islet insulitis. However, the majority of pancreas-infiltrating T cells accumulated around blood vessels in the exocrine pancreas. All T cell clones expressed the chemokine receptor CXCR3 that is associated with homing to insulitic lesions in men and mice.Conclusions/interpretation Our study provides the first evidence of in vivo accumulation in pancreatic tissue of islet-reactive T cells derived from type 1 diabetic patients. The fact that such T cells do not penetrate islets is in line with the concept that additional factors may be required for the entry of T cells into inflamed islets to become diabetogenic.     

Detection of Rh D-associated epitopes in human and animal tissues using human monoclonal anti-D antibodies

The distribution of Rh D-associated epitopes in human and animal tissues has been assessed by immunochemical techniques using six human monoclonal anti-D antibodies. The IgM antibody MAD-2 reacted predominantly with endothelial cells lining blood vessels and with animal leucocytes and human leucocytes from both Rh D-positive and Rh D-negative donors. Immunoblotting revealed reactivity with a 55 kDa tissue component which was most prominent in homogenates of heart, lung and spleen. In contrast, the IgG antibodies UCHD4 and FOG-1 stained smooth muscle, and UCHD4 in addition weakly stained cardiac and skeletal muscle and the glomeruli of kidney. Immunoblotting with UCHD4 revealed reactivity with a 27 kDa tissue component which was most prominent in heart homogenates. The results show that the six monoclonal anti-D antibodies recognize at least four different epitopes and that Rh D-associated epitopes may occur in non-erythroid cell types.     

A human anti-D monoclonal antibody selected for enhanced FcgammaRIII engagement clears RhD+ autologous red cells in human volunteers as efficiently as polyclonal anti-D antibodies

A human anti-RhD immunoglobulin G1 monoclonal antibody (mAb), R297, was tested in a phase I study to assess its ability to induce the clearance of antibody-coated autologous RhD⁺ red blood cells (RBCs) in healthy male volunteers. The clearance potency of R297 was compared with that of a marketed human polyclonal anti-D product (Rhophylac^®). This mAb has been selected for its ability to strongly engage Fc-gamma receptor IIIA and to mediate a potent antibody-dependent cell cytotoxicity (ADCC) against RhD⁺ RBCs. Autologous RhD⁺ RBCs were sensitized with either Rhophylac^® or R297 at three different coating percentages (25, 12·5 and 6·25%), before re-infusion. This phase I study showed that the human R297 mAb promoted rapid and complete clearance of RBCs, and showed activity that was at least as potent as the human polyclonal anti-D antibody preparation. Clearance of RBCs could still be observed when the percentage of R297 used to coat the RBCs was reduced to 6·25%. Finally, none of the adverse events was severe or considered to be related to R297. Thus, R297 is a promising candidate for the prevention of allo-immunization and represents a new generation of Fc-modified monoclonal antibodies with increased FcγRIII binding and increased ADCC.     

An in-vitro assessment of the functional activity of monoclonal anti-D

The response of human monocytes to red cells sensitized with known levels of monoclonal antibody to the Rh antigen D (anti-D) was compared with that of polyclonal anti-D. Monocyte response was determined by measuring red cell adherence, erythrophagocytosis, monocyte-mediated red cell lysis and luminol-dependent chemiluminescence. By all criteria, monoclonal and polyclonal antibodies showed comparable activity, with IgG3 antibodies promoting a greater monocyte-red cell interaction than IgG1 antibodies. It is suggested that monoclonal anti-D may be effective in the prophylaxis of haemolytic disease of the newborn, providing such material is clinically acceptable.     

Initial characterization of monoclonal antibodies against human monocytes.

Three monoclonal antibodies against human monocytes have been produced by somatic cell fusion. Extensive specificity analysis suggests that these antibodies react with most if not all human peripheral blood monocytes and not with highly purified T or B cells. Initial chemical characterization of the monocyte antigen recognized by two of these antibodies is presented. The molecule is a single polypeptide chain with an apparent molecular weight of 200,000. These reagents should prove useful in the clinical definition of disorders of monocyte differentiation, in studies of monocyte function, and in the elucidation of the genetics and structure of monocyte cell surface antigens.     

Development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice

BACKGROUND: Severe acute respiratory syndrome (SARS) remains a significant public health concern after the epidemic in 2003. Human monoclonal antibodies (MAbs) that neutralize SARS-associated coronavirus (SARS-CoV) could provide protection for exposed individuals. METHODS: Transgenic mice with human immunoglobulin genes were immunized with the recombinant major surface (S) glycoprotein ectodomain of SARS-CoV. Epitopes of 2 neutralizing MAbs derived from these mice were mapped and evaluated in a murine model of SARS-CoV infection. RESULTS: Both MAbs bound to S glycoprotein expressed on transfected cells but differed in their ability to block binding of S glycoprotein to Vero E6 cells. Immunoprecipitation analysis revealed 2 antibody-binding epitopes: one MAb (201) bound within the receptor-binding domain at aa 490-510, and the other MAb (68) bound externally to the domain at aa 130-150. Mice that received 40 mg/kg of either MAb prior to challenge with SARS-CoV were completely protected from virus replication in the lungs, and doses as low as 1.6 mg/kg offered significant protection. CONCLUSIONS: Two neutralizing epitopes were defined for MAbs to SARS-CoV S glycoprotein. Antibodies to both epitopes protected mice against SARS-CoV challenge. Clinical trials are planned to test MAb 201, a fully human MAb specific for the epitope within the receptor-binding region.     

Electrophysiological characterization of connexin 40 deficient hearts--in vivo studies in mice

Intercellular communication is not only mediated by extracellular transmitters, but also directly by gap junction channels. One channel is composed of two hexameric hemichannels which consist of six polypeptide subunits called connexines (Cx). In the mammalian heart the following connexines have been documented: Cx37, Cx40, Cx43, Cx45, Cx46, Cx50 and Cx57. The labeling by number represents the rounded, molecular mass of the amino acid sequences given in kD. If identical connexin-isotypes form both connexons of a gap junction channel, homotypic coupling exists and a homomeric gap junction channel is formed. Different connexin-isotypes within both connexons cause form heterotypic coupling and heteromeric gap junction channels. Each channel type has specific properties regarding permeability and electrical conductance. Beside a typical age-dependent alignment of gap junction channels on the surface of the cardiac myocytes, regional distribution of the different connexins is different at distinct parts of the mouse heart. Cx40 is not found in the ventricular working myocardium of mice. In the atria as well as in the conduction system, Cx40 is the most frequently expressed. In line with the localization and the conduction properties of distinct homotypic gap junction channels, the Cx40 deficient mouse is suitable for analysis of atrial arrhythmias. Cx40-deficiency in the mouse heart results in characteristic ECG changes like first degree atrioventricular block and prolongation of the QRS duration. Thus, an impairment of the sinuatrial, intraatrial and atrioventricular conduction properties is documented in Cx40 deficient mice. These observations are associated with an increased atrial vulnerability. The Cx40 deficient mouse provides a good example of the relevance of transgenic mouse models to clarify the mechanisms of arrhythmogenesis. The clinical impact of future transgenic mouse models depends on the cooperation of geneticists, basic researchers and clinicians.     

Functional characteristics of human T-cell subpopulations distinguished by a monoclonal antibody

In animals and in man, diverse immunologic functions are mediated by specialized T-cell (thymus-derived lymphocyte) subsets that are distinguishable from one another on the basis of differences in cell surface determinants. Unfortunately, in humans, subset-specific antibodies have been difficult to generate. In this study, production of a murine monoclonal antibody specific for a subset of human T cells was achieved by fusing a sensitized B cell (bone marrow-derived cell) with a myeloma cell and isolating the antibody secreted by the resultant hybrid clone. This antibody binds 30-35% of peripheral T lymphocytes (T_a⁺ cells) but fails to bind remaining T lymphocytes (T_a^- cells), B lymphocytes, or monocytes. T_a⁺ and T_a^- subpopulations were separated with a fluorescence-activated cell sorter and their in vitro responses to various stimuli were assessed. T_a⁺ and T_a^- cells respond equally well to soluble antigens, allogeneic B cells, and autologous B cells, but only T_a⁺ cells respond to concanavalin A. T_a⁺ cells cultured in the presence of concanavalin A gradually lose the T_a marker, an effect not observed after stimulation with phytohemagglutinin, soluble antigens, or alloantigens. These results suggest that the functional subpopulation of T cells defined by T_a does not correspond to any previously described human T cell subset. Furthermore, somatic cell hybridization has been shown to be a feasible method for production of monoclonal antibodies specific for subpopulations of human lymphocytes.     

Biochemical characterization of human myeloperoxidase using three specific monoclonal antibodies

We have developed three monoclonal antibodies (moAbs), MA1, MA3 and MB1, which react with different antigenic determinants of human myeloperoxidase (MPO). In MPO-positive culture cell lines, HL-60 and NKM1, analysis of MPO by pulse-chase experiments followed by immunoprecipitation with these moAbs revealed that MPO was composed of subunits of 59K, 18K and 14.8K dalton polypeptides which are plausibly derived from the 89 K precursor. MA1 and MB1 react with both the precursor and the mature forms of MPO. MA3 reacts with only the mature forms of MPO. Blocking experiments on MPO-related functions revealed that the three moAbs could be divided into two groups. MA1 and MA3 inhibit MPO activities such as tetraguaiacol formation, iodide oxidation and luminol-dependent chemiluminescence, while MB1 shows no such inhibition.     

Production and characterization of human monoclonal antibodies against Schistosoma mansoni

We have produced a panel of human monoclonal antibodies (MoAb) from patients infected with Schistosoma mansoni in order to analyse more carefully the human immune response to this helminth infection. This study describes the production, characterization and analysis of these MoAbs. Briefly, peripheral blood mononuclear cells from chronically infected patients were (1) isolated and stimulated with parasite antigens in vitro, (2) positively selected for B-cells on anti-Ig columns, and (3) then transformed with Epstein-Barr virus (EBV). Once EBV cell lines were established, they were selected for anti-S. mansoni antibodies using an ELISA, cloned, retested and then fused with the mouse-human heteromyeloma SHM-D33. In this study, we describe five MoAbs which have different antigenic specificities for life-cycle stages based on ELISA to soluble crude antigen preparations, membrane immunofluorescence on whole intact organisms, and immunofluorescent staining of cryostat frozen sections. The importance of these reagents with regard to the human immune response to S. mansoni is currently being evaluated.     

In vivo stabilization of mutant human transthyretin in transgenic mice

Transthyretin (TTR) is a 55 kD homotetrameric serum protein transporter of retinol binding protein charged with retinol and thyroxine (T4). The highly amyloidogenic human TTR variant in which leucine at position 55 is replaced by proline (L55P TTR) is responsible for aggressive fatal amyloidosis with peripheral and autonomic neuropathy, cardiomyopathy and nephropathy. Mice bearing one or two copies of a 19.2 kB human genomic fragment containing the entire coding sequence and the known control regions of the L55P TTR transgene, failed to develop TTR amyloidosis even though their sera contained mutant human TTR. The frequency of TTR tissue deposition was increased when the L55P TTR transgene was bred onto a murine TTR-null background. Denaturation of sera from the transgenic animals and murine TTR-knockouts expressing the human L55P TTR transgene revealed that the TTR tetramer was much more stable in the presence of the murine protein because the TTR circulates as hybrid human/murine heterotetramers. Intraperitoneal administration of diflunisal, a non-steroidal anti-inflammatory drug that binds to TTR in its T4-binding site and inhibits fibril formation in vitro, to human L55P TTR transgenic animals in which the murine TTR gene had been silenced, also stabilizes the circulating mutant protein to in vitro urea denaturation.     

Functional characterization of human thyroid tissue with immunohistochemistry.

Immunohistochemistry provides insights in the expression of functional proteins and of their localization in normal thyroid tissue and in thyroid diseases. In hyperfunctional thyroid tissues, staining for sodium/iodide symporter (NIS), pendrin, thyroid peroxidase (TPO), and thyroglobulin (Tg) is increased. In hypofunctioning thyroid tissues, NIS staining is markedly decreased; in benign hypofunctioning adenomas, the expression of the other functional proteins is unmodified or slightly decreased, whereas their expression is profoundly decreased or absent in differentiated thyroid carcinoma.     

In vivo ultrasonic tissue characterization of human intracardiac masses

Interactions between an ultrasonic signal and cardiac tissue have been used to characterize the histologic state of myocardium in vitro. To assess the utility of in vivo ultrasonic tissue characterization, stochastic analysis was applied to the digitized echocardiographic signals from 15 patients with 2-dimensional echocardiograms suggesting intracardiac masses. Ten subjects with echocardiograms suggesting mural thrombi underwent subsequent surgery or necropsy, which confirmed thrombi in 6 and revealed no thrombi (designated artifact) in 4. Five other patients had intracardiac tumors. The amplitudes within the digitized ultrasonic signals were displayed as histograms, which were described by a parameter k that represented the degree to which each histogram departed from a totally random probability density function. In 5 of 6 thrombi, k = 0, but in all 4 artifacts, k greater than 0. The sixth thrombus had k = 0.5 due to the specular effect of the interface between the thrombus' 2 lobes. All 5 tumors had k greater than 0. Ultrasonic tissue characterization using a stochastic analysis of backscatter can be performed in vivo and helps differentiate thrombus from artifact and tumor in the heart.     

A human monoclonal antibody that protects mice against Pseudomonas-induced pneumonia

X-linked immunodeficient (xid) (CBA/N female x DBA/2 male) F1 male mice, when treated with cyclophosphamide, were much more susceptible to challenge with aerosolized Pseudomonas aeruginosa serotype 11 than were control F1 female littermates. Mortality of F1 males was decreased significantly after intravenous administration of human P. aeruginosa serotype 11 O-specific monoclonal antibody. Antibody treatment reduced bacterial titers in the lungs as well as the severity of Pseudomonas-induced lung histopathology.     

Production and characterization of human monoclonal anti-idiotype antibodies to anti-dsDNA antibodies

Anti-dsDNA autoantibodies are the hallmark of systemic lupus erythematosus (SLE) and frequently correlate with disease activity. In this study we report the isolation and characterization of human anti-Id monoclonal antibody fragments as single-chain Fv fragments (scFv) against anti-dsDNA antibody. The anti-Id monoclonal antibodies, specific for anti-dsDNA antibodies, have been cloned from phage display antibody scFv libraries derived from a patient with SLE. The V gene repertoires were derived from the RNA obtained from the B cells of an SLE patient with anti-Ro/SSA and anti-La/SSB antibodies. Affinity-purified anti-dsDNA antibodies were used for selection of bacterial clones producing specific scFv antibody fragments against anti-dsDNA antibodies and little reactivity with normal IgG and other IgG antibodies by ELISA. The anti-Id antibody recognizes a public idiotope that is broadly cross-reactive with polyclonal and monoclonal anti-dsDNA antibodies. This binding was largely inhibited by dsDNA antigen. The anti-Id antibody inhibited anti-dsDNA binding to dsDNA antigen in immunoassays and in the Crithidia luciliae assay. The anti-Id scFv antibody fragments derived from human genes could modulate the pathogenicity of anti-dsDNA autoantibodies and may have therapeutic implications in SLE. They may also be used as probes in studies of the structure of the idiotype.