Immunoglobulin heavy chain sequence analysis in Waldenstrom's macroglobulinemia and immunoglobulin M monoclonal gammopathy of undetermined significance.

In this study, we used IGH sequence analysis to assess the maturational status of Waldenstrom's (WM) macroglobulinemia and its putative precursor immunoglobulin (Ig)-M monoclonal gammopathy of undetermined significance (MGUS). IGH sequence analysis was performed using standard methods in 23 cases (20 WM and 3 IgM MGUS as defined by consensus panel criteria). Waldenstrom's macroglobulinemia cases were characterized by heavily mutated IGH genes (median, 6.3%; range, 3.8%-13.9%) but without intraclonal variation (ICV). IgM MGUS was similarly characterized by somatic hypermutation (median, 7.5%; range, 7%-7.7%), but ICV was evident in 1 of the 3 cases. We would therefore conclude that WM is characterized by somatic hypermutation without ICV, which supports a derivation from postgerminal center/memory B cells. IgM MGUS is also characterized by somatic hypermutation but, in a manner similar to IgA/IgG MGUS, can be associated with ICV, although the significance of this remains unclear.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma.

BACKGROUND AND OBJECTIVE: Molecular analysis by PCR of monoclonally rearranged immunoglobulin (Ig) genes can be used for diagnosis in B-cell lymphoproliferative disorders (LPD), as well as for monitoring minimal residual disease (MRD) after treatment. This technique has the risk of false-positive results due to the "background" amplification of similar rearrangements derived from polyclonal B-cells. This problem can be resolved in advance by additional analyses that discern between polyclonal and monoclonal PCR products, such as the heteroduplex analysis. A second problem is that PCR frequently fails to amplify the junction regions, mainly due to somatic mutations frequently present in mature (post-follicular) B-cell lymphoproliferations. The use of additional targets (e.g. Ig light chain genes) can avoid this problem. DESIGN AND METHODS: We studied the specificity of heteroduplex PCR analysis of several Ig junction regions to detect monoclonal products in samples from 84 MM patients and 24 patients with B cell polyclonal disorders. RESULTS: Using two distinct VH consensus primers (FR3 and FR2) in combination with one JH primer, 79% of the MM displayed monoclonal products. The percentage of positive cases was increased by amplification of the Vlamda-Jlamda junction regions or kappa(de) rearrangements, using two or five pairs of consensus primers, respectively. After including these targets in the heteroduplex PCR analysis, 93% of MM cases displayed monoclonal products. None of the polyclonal samples analyzed resulted in monoclonal products. Dilution experiments showed that monoclonal rearrangements could be detected with a sensitivity of at least 10(-2) in a background with >30% polyclonal B-cells, the sensitivity increasing up to 10(-3) when the polyclonal background was

Conditioning film and environmental effects on the adherence of Candida spp. to silicone and poly(vinylchloride) biomaterials.

The reported incidence of colonization of oropharyngeal medical devices with Candida spp. has increased in recent years, although few studies that have systematically examined the adherence of yeast cells to such biomaterials, the primary step in the process of colonization. This study, therefore, examined the effects of oropharyngeal atmospheric conditions (5% v/v carbon dioxide) and the presence of a salivary conditioning film on both the surface properties and adherence of Candida albicans, Candida krusei and Candida tropicalis to PVC and silicone. Furthermore, the effects of the salivary conditioning film on the surface properties of these biomaterials are reported. Growth of the three Candida spp. in an atmosphere containing 5% v/v CO2 significantly increased their cell surface hydrophobicity and reduced the zeta potential of C. albicans and C. krusei yet increased the zeta potential of C. tropicalis (p < 0.05). Furthermore, growth in 5% v/v CO2 decreased the adherence of C. tropicalis and C. albicans to both PVC and silicone, however, increased adherence of C. krusei (p < 0.05). Pre-treatment of the microorganisms with pooled human saliva significantly decreased their cell surface hydrophobicity and increased their adherence to either biomaterial in comparison to yeast cells that had been pre-treated with PBS (p < 0.05). Saliva treatment of the microorganisms had no consistent effect on microbial zeta potential. Interestingly, adherence of the three, saliva-treated Candida spp. to saliva-treated silicone and PVC was significantly lower than whenever the microorganisms and biomaterials had been treated with PBS (p < 0.05). Treatment of silicone and PVC with saliva significantly altered the surface properties, notably reducing both the advancing and receding contact angles and, additionally, the microrugosity. These effects may contribute to the decreased adherence of saliva-treated microorganisms to these biomaterials. In conclusion, this study has demonstrated the effects of physiological conditions within the oral cavity on the adherence of selected Candida spp. to biomaterials employed as oropharyngeal medical devices. In particular, this study has ominously shown that these materials act as substrates for yeast colonization, highlighting the need for advancements in biomaterial design. Furthermore, it is important that physiological conditions should be employed whenever biocompatibility of oropharyngeal biomaterials is under investigation. © 2001 Kluwer Academic Publishers.

CD33 reponses are blocked by SOCS3 through accelerated proteasomal-mediated turnover

CD33 is a member of the sialic acid–binding immunoglobulin-like lectin (Siglec) family of inhibitory receptors and a therapeutic target for acute myeloid leukemia (AML). CD33 contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), which can recruit SHP-1 and SHP-2. How CD33 expression is regulated is unclear. Suppressor of cytokine signaling 3 (SOCS3) is expressed in response to cytokines, LPS, and other PAMPs, and competes with SHP-1/2 binding to ITIMs of cytokine receptors, thereby inhibiting signaling. In this study, using peptide pull-down experiments, we found that SOCS3 can specifically bind to the phosphorylated ITIM of CD33. Additionally, following cross-linking SOCS3 can recruit the ECS E3 ligase resulting in accelerated proteasomal degradation of both CD33 and SOCS3. Our data suggest that the tyrosine motifs in CD33 are not important for internalization, while they are required for degradation. Moreover, SOCS3 inhibited the CD33-induced block on cytokine-induced proliferation. This is the first receptor shown to be degraded by SOCS3 and where SOCS3 and its target protein are degraded concomitantly. Our findings clearly suggest that during an inflammatory response, the inhibitory receptor CD33 is lost by this mechanism. Moreover, this has important clinical implications as tumors expressing SOCS3 may be refractory to -CD33 therapy.

SOCS3 Targets Siglec 7 for Proteasomal Degradation and Blocks Siglec 7-mediated Responses

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5–11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.

Impaired retinal angiogenesis in diabetes: role of advanced glycation end products and galectin-3

Suppression of angiogenesis during diabetes is a recognized phenomenon but is less appreciated within the context of diabetic retinopathy. The current study has investigated regulation of retinal angiogenesis by diabetic serum and determined if advanced glycation end products (AGEs) could modulate this response, possibly via AGE-receptor interactions. A novel in vitro model of retinal angiogenesis was developed and the ability of diabetic sera to regulate this process was quantified. AGE-modified serum albumin was prepared according to a range of protocols, and these were also analyzed along with neutralization of the AGE receptors galectin-3 and RAGE. Retinal ischemia and neovascularization were also studied in a murine model of oxygen-induced proliferative retinopathy (OIR) in wild-type and galectin-3 knockout mice (gal3(-/-)) after perfusion of preformed AGEs. Serum from nondiabetic patients showed significantly more angiogenic potential than diabetic serum (P <0.0001) and within the diabetic group, poor glycemic control resulted in more AGEs but less angiogenic potential than tight control (P <0.01). AGE-modified albumin caused a dose-dependent inhibition of angiogenesis (P <0.001), and AGE receptor neutralization significantly reversed the AGE-mediated suppression of angiogenesis (P <0.01). AGE-treated wild-type mice showed a significant increase in inner retinal ischemia and a reduction in neovascularization compared with non-AGE controls (P <0.001). However, ablation of galectin-3 abolished the AGE-mediated increase in retinal ischemia and restored the neovascular response to that seen in controls. The data suggest a significant suppression of angiogenesis by the retinal microvasculature during diabetes and implicate AGEs and AGE-receptor interactions in its causation.

T-cell-rich histiocyte-rich B-cell lymphoma of parotid gland

We describe a malignant lymphoma of the parotid gland arising in a patient with Sjögren's syndrome. Diagnosis was established on needle biopsy which showed a mixed population of lymphoid cells. Immunohistochemistry revealed B-lymphoid cells, T-lymphoid cells and histiocytes. Clonal immunoglobulin heavy chain gene rearrangement was demonstrated using the polymerase chain reaction. Within the confines of the small biopsy, the lesion qualifies for the designation T-cell-rich histiocyte-rich B-cell lymphoma. The value of molecular techniques in the diagnosis of malignant lymphoma on limited tissue samples is highlighted by this case.

The mouse and human IGSF6 (DORA) genes map to the inflammatory bowel disease 1 locus and are embedded in an intron of a gene of unknown function.

We have previously characterized IGSF6 (DORA), a novel member of the immunoglobulin superfamily (IGSF) from human and rat expressed in dendritic and myeloid cells. Using a probe from the open reading frame of the rat cDNA, we isolated a cosmid which contains the entire mouse gene. By comparative analysis and reverse transcriptase polymerase chain reaction, we defined the intron/exon structure and the mRNA of the mouse gene and, with respect to human BAC clones, the human gene. The genes span 10 kb (mouse) and 12 kb (human), with six exons arranged in a manner similar to other members of the IGSF. All intron/exon boundaries follow the GT-AG rule. Expression of the mouse Igsf6 gene is restricted to cells of the immune system, particularly macrophages. Northern blot revealed a single mRNA of 2.5 kb, in contrast to the human gene which is expressed as two mRNAs of 1 and 2.5 kb. The human and mouse genes were localized to a locus associated with inflammatory bowel disease. Analysis of the flanking regions of the Igsf6 gene revealed the presence of an unrelated gene, transcribed from the opposite strand of the DNA and oriented such that the Igsf6 gene is encoded entirely within an intron. An identical organization is seen in human. This gene of unknown function is transcribed and processed, contains homologues in Caenorhabditis elegans and prokaryotes, and is expressed in most organs in the mouse.

Inflammatory mediators in bronchoalveolar lavage samples from children with and without asthma

Background: We investigated whether eosinophils and mast cells, found in the airways of children with wheeze, were activated during relatively asymptomatic periods.
Methods: A nonbronchoscopic bronchoalveolar lavage (BAL) procedure was performed on children presenting for an elective surgical procedure. Eosinophil-derived (eosinophil cationic protein, ECP) and mast cell-derived (histamine/tryptase) mediator concentrations were measured in the BAL fluid. A detailed history and serum immunoglobulin E were used to classify the children into four groups: atopic with and without asthma, viral-associated wheeze and normal controls.
Results: The ECP concentrations in BAL from atopic asthmatic subjects were significantly higher than those measured in BAL from normal controls (P < 0.01), no other groups differed significantly. Histamine concentrations were elevated in both the atopic asthmatic and viral-associated wheeze groups compared with controls (P < 0.02) and additionally higher concentrations were obtained in atopics with asthma compared with atopics without asthma (P < 0.03). Tryptase concentrations did not differ between groups, although the tryptase and histamine concentrations correlated significantly (r = 0.78, P < 0.0001).
Conclusions: Elevated histamine concentrations were found in children with wheeze regardless of the aetiology, whereas ECP was only elevated in those asthmatics with atopy. This suggests that even in relatively quiescent periods, there is some on going activation of airway eosinophils in children with atopic asthma.

Interleukin-4 and interleukin-4 soluble receptor α levels in bronchoalveolar lavage from children with asthma

Background: In asthma there is increased expression of the Th2-type cytokine interleukin-4 (IL-4). IL-4 is important in immunoglobulin isotype switching to immunoglobulin E and adhesion of eosinophils to endothelium.

Objectives: We hypothesized that levels of IL-4 in bronchoalveolar lavage (BAL) fluid would be increased in stable, atopic asthmatic children compared with controls and that levels of its physiologic inhibitor IL-4 soluble receptor α (IL-4sRα) would be correspondingly decreased.

Methods: One hundred sixteen children attending a children's hospital for elective surgery were recruited. A nonbronchoscopic BAL was performed, and IL-4 and IL-4sRα were measured in the BAL supernatants.

Results: There was no significant difference in IL-4 concentrations between atopic asthmatic children, atopic normal controls, and nonatopic normal controls [0.13 pg/mL (0.13 to 0.87) vs 0.13 pg/mL (0.13 to 0.41) vs 0.13 pg/mL (0.13 to 0.5), P = 0.65]. IL-4sRα levels were significantly increased in asthmatic patients compared with atopic controls [6.4 pg/mL (5.0 to 25.5) vs 5.0 pg/mL (5.0 to 9.9), P = 0.018], but not when compared with the nonatopic controls [5.2 pg/mL (5.0 to 10.6), P = 0.19].

Conclusions: Contrary to expectation, IL-4sRα levels are increased in BAL from stable asthmatic children compared with nonatopic controls, and we speculate that IL-4sRα is released by inflammatory cells in the airways to limit the proinflammatory effects of IL-4.

Enhanced lymphocyte interferon (IFN)-γ responses in a PTEN mutation-negative Cowden disease kindred.

Identification of immune modifiers of inherited cancer syndromes may provide a rationale for preventive therapy. Cowden disease (CD) is a genetically heterogeneous inherited cancer syndrome that arises predominantly from germline phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation and increased phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signalling. However, many patients with classic CD diagnostic features are mutation-negative for PTEN (PTEN M-Neg). Interferon (IFN)-gamma can modulate the PI3K/mTOR pathway, but its association with PTEN M-Neg CD remains unclear. This study assessed IFN-gamma secretion by multi-colour flow cytometry in a CD kindred that was mutation-negative for PTEN and other known susceptibility genes. Because IFN-gamma responses may be regulated by killer cell immunoglobulin-like receptors (KIR) and respective human leucocyte antigen (HLA) ligands, KIR/HLA genotypes were also assessed. Activating treatments induced greater IFN-gamma secretion in PTEN M-Neg CD peripheral blood lymphocytes versus healthy controls. Increased frequency of activating KIR genes, potentially activating KIR/HLA compound genotypes and reduced frequency of inhibitory genotypes, were found in the PTEN M-Neg CD kindred. Differences of IFN-gamma secretion were observed among PTEN M-Neg CD patients with distinct KIR/HLA compound genotypes. Taken together, these findings show enhanced lymphocyte secretion of IFN-gamma that may influence the PI3K/mTOR CD causal molecular pathway in a PTEN mutation-negative CD kindred.

PI3K beta Plays a Critical Role in Neutrophil Activation by Immune Complexes

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (Fc gamma Rs). Here, we used genetic and pharmacological approaches to define a selective role for the beta isoform of phosphoinositide 3-kinase (PI3K beta) in Fc gamma R-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3K beta alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3K beta and PI3K delta, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3K beta by immune complexes involved cooperation between Fc gamma Rs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B-4. Coincident activation by a tyrosine kinase-coupled receptor (Fc gamma R) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the beta isoform of PI3K. PI3K beta-deficient mice were highly protected in an Fc gamma R-dependent model of autoantibody-induced skin blistering and were partially protected in an Fc gamma R-dependent model of inflammatory arthritis, whereas combined deficiency of PI3K beta and PI3K delta resulted in near-complete protection in the latter case. These results define PI3K beta as a potential therapeutic target in inflammatory disease.

Comparison of techniques to screen and characterize bacteria-specific hybridomas for high-quality monoclonal antibodies selection

Antibodies are are very important materials for diagnostics. A rapid and simple hybridoma screening method will help in delivering specific monoclonal antibodies. In this study, we systematically developed the first antibody array to screen for bacteria-specific monoclonal antibodies using Listeria monocytogenes as a bacteria model. The antibody array was developed to expedite the hybridoma screening process by printing hybridoma supernatants on a glass slide coated with an antigen of interest. This screening method is based on the binding ability of supernatants to the coated antigen. The bound supernatants were detected by a fluorescently labeled anti-mouse immunoglobulin. Conditions (slide types, coating, spotting, and blocking buffers) for antibody array construction were optimized. To demonstrate its usefulness, antibody array was used to screen a sample set of 96 hybridoma supernatants in comparison to ELISA. Most of the positive results identified by ELISA and antibody array methods were in agreement except for those with low signals that were undetectable by antibody array. Hybridoma supernatants were further characterized with surface plasmon resonance to obtain additional data on the characteristics of each selected clone. While the antibody array was slightly less sensitive than ELISA, a much faster and lower cost procedure to screen clones against multiple antigens has been demonstrated. (C) 2011 Elsevier Inc. All rights reserved.

INTRAVASCULAR ANTI-IGE CHALLENGE IN PERFUSED LUNGS - MEDIATOR RELEASE AND VASCULAR PRESSOR-RESPONSE

Intravascular application of goat anti-rabbit immunoglobulin E (IgE) was used to stimulate parenchymal mast cells in situ in perfused rabbit lungs. Sustained pulmonary arterial pressure rise was evoked in the absence of lung vascular permeability increase and lung edema formation. Early prostaglandin (PG) D2 and histamine release into the perfusate was documented, accompanied by more sustained liberation of cysteinyl leukotrienes (LT), LTB4, and PGI2. The quantities of these inflammatory mediators displayed the following order: histamine > cysteinyl-LT > PGI2 > LTB4 > PGD2. Pressor response and inflammatory mediator release revealed corresponding bell-shaped dose dependencies. Cyclooxygenase inhibition (acetylsalicylic acid) suppressed prostanoid generation, increased LT release, and did not substantially affect pressor response and histamine liberation. BW755 C, a cyclo- and lipoxygenase inhibitor, blocked the release of cysteinyl-LT and markedly reduced the liberation of the other inflammatory mediators as well as the pressor response. The H-1-antagonist clemastine caused a moderate reduction of the anti-IgE-provoked pressure rise. We conclude that intravascular anti-IgE challenge in intact lungs provokes the release of an inflammatory mediator profile compatible with in situ lung parenchymal mast cell activation. Pulmonary hypertension represents the predominant vascular response, presumably mediated by cysteinyl-LT and, to a minor extent, histamine liberation.