Cancer-associated V-ATPase induces delayed apoptosis of protumorigenic neutrophils.

Tumors and neutrophils undergo an unexpected interaction, in which products released by tumor cells interact to support neutrophils that in turn support cancer growth, angiogenesis, and metastasis. A key protein that is highly expressed by cancer cells in tumors is the a2 isoform V-ATPase (a2V). A peptide from a2V (a2NTD) is secreted specifically by cancer cells, but not normal cells, into the tumor microenvironment. This peptide reprograms neutrophils to promote angiogenesis, cancer cell invasiveness, and neutrophil recruitment. Here, we provide evidence that cancer-associated a2V regulates the life span of protumorigenic neutrophils by influencing the intrinsic pathway of apoptosis. Immunohistochemical analysis of human cancer tissue sections collected from four different organs shows that levels of a2NTD and neutrophil counts are increased in cancer compared with normal tissues. Significant increases in neutrophil counts were present in both poorly and moderately differentiated tumors. In addition, there is a positive correlation between the number of neutrophils and a2NTD expression. Human neutrophils treated with recombinant a2NTD show significantly delayed apoptosis, and such prolonged survival was dependent on NF-κB activation and ROS generation. Induction of antiapoptotic protein expression (Bcl-xL and Bcl-2A1) and decreased expression of proapoptotic proteins (Bax, Apaf-1, caspase-3, caspase-6, and caspase-7) were a hallmark of these treated neutrophils. Autocrine secretion of prosurvival cytokines of TNF-α and IL-8 by treated neutrophils prolongs their survival. Our findings highlight the important role of cancer-associated a2V in regulating protumorigenic innate immunity, identifying a2V as a potential important target for cancer therapy.

Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of Autophagy, Endosome Function, and ERK/MEK Pathway.

Ovarian cancer (OVCA) patients often develop tolerance to standard platinum therapy that accounts for extensive treatment failures. Cisplatin resistant OVCA cells (cis-R) display enhanced survival mechanisms to cope with therapeutic stress. In these cells, increased autophagy process assists in chemoresistance by boosting the nutrient pool under stress. To improve the treatment response, both protective autophagy inhibition and its overactivation are showing efficacy in chemosensitization. Autophagy requires a tightly regulated intracellular pH. Vacuolar ATPases (V-ATPases) are proton extruding nanomotors present on cellular/vesicular membranes where they act as primary pH regulators. V-ATPase 'a2' isoform (V0a2), the major pH sensing unit, is markedly overexpressed on the plasma membrane and the early endosomes of OVCA cells. Previously, V0a2 inhibition sensitized cis-R cells to platinum drugs by acidifying cytosolic pH that elevated DNA damage. Here, we examined how V0a2 inhibition affected endosomal function and the autophagy process as a possible factor for cisplatin sensitization. Clinically, V0a2 expression was significantly higher in tissues from drug nonresponder OVCA patients compared to treatment responders. In vitro V0a2 knockdown in cis-R cells (sh-V0a2-cisR) significantly reduced the tumor sphere-forming ability and caused complete disintegration of the spheres upon cisplatin treatment. The apoptotic capacity of sh-V0a2-cisR improved substantially with potentiation of both intrinsic and extrinsic apoptotic pathway when treated with cisplatin. Unlike the chemical V-ATPase inhibitors that acutely induce autophagy, here, the stable V0a2 inhibition dampened the protective autophagy process in sh-V0a2-cisR cells with downregulated expression of proteins beclin-1, ATG-7, and LC3B and low autophagosome numbers compared to control cis-R cells. These cells showed downregulated ERK/MEK pathway that is known to repress autophagy. Interestingly, upon cisplatin treatment of sh-V0a2-cisR, the autophagy initiation proteins (LC3B, ATG7, and Beclin 1) were found upregulated as a stress response compared to the untreated cells. However, there was a concomitant downstream autophagosome accumulation and an enhanced P62 protein levels indicating the overall block in autophagy flux. Mechanistically, V0a2 knockdown caused defects in early endosome function as the transferrin internalization was impaired. Taken together, this study provides a novel insight into the mechanism by which V-ATPase-isoform regulates autophagy that assists in chemoresistance in ovarian cancer. We conclude that V-ATPase-V0a2 is a potent target for developing an effective treatment to enhance patient survival rates in ovarian cancer.

Hematopoietic stem cell specific V-ATPase controls breast cancer progression and metastasis via cytotoxic T cells.

The interaction of recruited immune effector cells and cancer cells within tumor microenvironment (TME) shapes the fate of cancer progression and metastasis. Many cancers including breast cancer, express a specific vacuolar ATPase (a2V) on their cell surface which acidifies the extracellular milieu helping cancer cell proliferation and metastasis. To understand the role of immune cell-associated-a2V during breast tumor pathogenesis, we knocked-out a2V (KO) from the hematopoietic stem cells (HSC) and generated breast tumors in mice. The a2V-KO mice developed faster growing, larger, and metastatic breast tumors compared to control mice. Further investigation of the TME revealed a significant reduction in the presence of CD4+ and CD8+ T cells in the a2V-KO tumors. Targeted RNA-Seq of the cells of the TME demonstrated that pro-inflammatory cytokines, death receptors, death receptor ligands, and cytotoxic effectors were significantly down-regulated within the a2V-KO TME. Interestingly, analysis of immune cells in the blood, spleen, and thymus of the non-tumor bearing a2V-KO mice revealed a significant decrease in CD4+ and CD8+ T cell populations. For the first time, this study demonstrates that inhibition of V-ATPase expression in HSC leads to a decrease in CD4+ and CD8+ T cell populations and thus promotes breast tumor growth and metastasis.

Caspase-11-dependent pyroptosis of lung epithelial cells protects from melioidosis while caspase-1 mediates macrophage pyroptosis and production of IL-18.

Infection with Burkholderia pseudomallei or B. thailandensis triggers activation of the NLRP3 and NLRC4 inflammasomes leading to release of IL-1ß and IL-18 and death of infected macrophages by pyroptosis, respectively. The non-canonical inflammasome composed of caspase-11 is also activated by these bacteria and provides protection through induction of pyroptosis. The recent generation of bona fide caspase-1-deficient mice allowed us to reexamine in a mouse model of pneumonic melioidosis the role of caspase-1 independently of caspase-11 (that was also absent in previously generated Casp1-/- mice). Mice lacking either caspase-1 or caspase-11 were significantly more susceptible than wild type mice to intranasal infection with B. thailandensis. Absence of caspase-1 completely abolished production of IL-1ß and IL-18 as well as pyroptosis of infected macrophages. In contrast, in mice lacking caspase-11 IL-1ß and IL-18 were produced at normal level and macrophages pyroptosis was only marginally affected. Adoptive transfer of bone marrow indicated that caspase-11 exerted its protective action both in myeloid cells and in radio-resistant cell types. B. thailandensis was shown to readily infect mouse lung epithelial cells triggering pyroptosis in a caspase-11-dependent way in vitro and in vivo. Importantly, we show that lung epithelial cells do not express inflammasomes components or caspase-1 suggesting that this cell type relies exclusively on caspase-11 for undergoing cell death in response to bacterial infection. Finally, we show that IL-18's protective action in melioidosis was completely dependent on its ability to induce IFNγ production. In turn, protection conferred by IFNγ against melioidosis was dependent on generation of ROS through the NADPH oxidase but independent of induction of caspase-11. Altogether, our results identify two non-redundant protective roles for caspase-1 and caspase-11 in melioidosis: Caspase-1 primarily controls pyroptosis of infected macrophages and production of IL-18. In contrast, caspase-11 mediates pyroptosis of infected lung epithelial cells.

Mammary epithelium-specific inactivation of V-ATPase reduces stiffness of extracellular matrix and enhances metastasis of breast cancer.

Extracellular matrix (ECM) critically impacts tumor progression and is influenced by both cancer and host tissue cells. While our understanding of cancer cell ECM remodeling is widespread, the importance of host tissue ECM, which provides initial congenial environment for primary tumor formation, is partly understood. Here, we report a novel role of epithelial cell-associated vacuolar ATPase 'a2' isoform (a2V) in regulating breast tissue ECM stiffness to control metastasis. Using a mammary gland-specific a2V-knockout model, we show that in the absence of a2V, breast tumors exhibit atypically soft tumor phenotype, less tumor rigidity, and necrotic tumor microenvironment. These tumors contain a decreased number of cancer cells at primary tumor site, but showed extensive metastases compared to control. Nanomechanical evaluation of normal breast tissues revealed a decrease in stiffness and collagen content in ECM of a2V-deleted breast tissues. Mechanistically, inhibition of a2V expression caused dispersed Golgi morphology with relocation of glycosyltransferase enzymes to early endosomes in mammary epithelial cells. This resulted in defective glycosylation of ECM proteins and production of compromised ECM that further influenced tumor metastasis. Clinically, in patients with cancer, low a2V expression levels in normal breast tissue correlated with lymph node metastasis. Thus, using a new knockout mouse model, we have identified a2V expression in epithelial cells as a key requirement for proper ECM formation in breast tissue and its expression levels can significantly modulate breast tumor dissemination. Evaluation of a2V expression in normal breast tissues can help in identifying patients with high risk of developing metastases.

Role of Canonical and Non-canonical Inflammasomes During Burkholderia Infection.

Burkholderia pseudomallei is a Gram-negative flagellate bacterium that causes melioidosis, a disease endemic to Southeast Asia and other tropical regions. Following infection of macrophages and other non-phagocytic cell types, B. pseudomallei or B. thailandensis (a related species that causes disease in mice but not humans) are able to escape the phagosome and replicate in the host cell cytoplasm. Resistance to infection with Burkholderia is dependent on the Nlrp3 and Nlrc4 inflammasomes and the non-canonical caspase-11 inflammasome. Nlrc4 mediates protection through induction of pyroptosis in the early phase of infection. As the infection progresses and as IL-18-dependent IFNγ production increases, caspase-11-dependent pyroptosis acquires a preponderant protective role. Production of IL-1ß and IL-18 during infection is primarily mediated by Nlrp3. IL-18 is essential for survival because of its ability to induce IFNγ production, which in turn activates macrophage microbicidal functions and primes for caspase-11 expression. In contrast, during melioidosis, IL-1ß has deleterious effects due to excessive recruitment of neutrophils to the lung and consequent tissue damage.

Aortopulmonary Window With Anomalous Coronary Arteries.

Aortopulmonary window associated with anomalous origin of the right coronary artery from the main pulmonary artery is rare. We report a four-month-old male presenting with anomalous origin of both right and left coronary arteries from a single ostium from the anterior sinus of the pulmonary artery along with aortopulmonary window (APW). The patient was managed successfully with a pericardial baffle shunting the coronary ostium to the aorta through the APW.

Production of anti-LPS IgM by B1a B cells depends on IL-1ß and is protective against lung infection with Francisella tularensis LVS.

The role of IL-1ß and IL-18 during lung infection with the gram-negative bacterium Francisella tularensis LVS has not been characterized in detail. Here, using a mouse model of pneumonic tularemia, we show that both cytokines are protective, but through different mechanisms. Il-18-/- mice quickly succumb to the infection and showed higher bacterial burden in organs and lower level of IFNγ in BALF and serum compared to wild type C57BL/6J mice. Administration of IFNγ rescued the survival of Il-18-/- mice, suggesting that their decreased resistance to tularemia is due to inability to produce IFNγ. In contrast, mice lacking IL-1 receptor or IL-1ß, but not IL-1α, appeared to control the infection in its early stages, but eventually succumbed. IFNγ administration had no effect on Il-1r1-/- mice survival. Rather, Il-1r1-/- mice were found to have significantly reduced titer of Ft LPS-specific IgM. The anti-Ft LPS IgM was generated in a IL-1ß-, TLR2-, and ASC-dependent fashion, promoted bacteria agglutination and phagocytosis, and was protective in passive immunization experiments. B1a B cells produced the anti-Ft LPS IgM and these cells were significantly decreased in the spleen and peritoneal cavity of infected Il-1b-/- mice, compared to C57BL/6J mice. Collectively, our results show that IL-1ß and IL-18 activate non-redundant protective responses against tularemia and identify an essential role for IL-1ß in the rapid generation of pathogen-specific IgM by B1a B cells.

Neutrophil elastase causes tissue damage that decreases host tolerance to lung infection with burkholderia species.

Two distinct defense strategies can protect the host from infection: resistance is the ability to destroy the infectious agent, and tolerance is the ability to withstand infection by minimizing the negative impact it has on the host's health without directly affecting pathogen burden. Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and causes melioidosis. We have recently shown that inflammasome-triggered pyroptosis and IL-18 are equally important for resistance to B. pseudomallei, whereas IL-1ß is deleterious. Here we show that the detrimental role of IL-1ß during infection with B. pseudomallei (and closely related B. thailandensis) is due to excessive recruitment of neutrophils to the lung and consequent tissue damage. Mice deficient in the potentially damaging enzyme neutrophil elastase were less susceptible than the wild type C57BL/6J mice to infection, although the bacterial burdens in organs and the extent of inflammation were comparable between C57BL/6J and elastase-deficient mice. In contrast, lung tissue damage and vascular leakage were drastically reduced in elastase-deficient mice compared to controls. Bradykinin levels were higher in C57BL/6 than in elastase-deficient mice; administration of a bradykinin antagonist protected mice from infection, suggesting that increased vascular permeability mediated by bradykinin is one of the mechanisms through which elastase decreases host tolerance to melioidosis. Collectively, these results demonstrate that absence of neutrophil elastase increases host tolerance, rather than resistance, to infection by minimizing host tissue damage.

Cold agglutinin disease detected during open heart surgery.

Cold agglutinins are commonly found in sera of healthy persons. They rarely become clinically apparent due to their activity at lower temperature. In these patients, cardio-vascular operations requiring hypothermia can result in complications like hemolysis, renal failure, myocardial damage and cause unexpected morbidity and even mortality (Agarwal et al., Ann Thorac Surg 60:1143-1150, 1995). Ideally, all patients should be routinely screened pre-operatively for antibodies and management plans made accordingly but the low incidence of the disease, cost of screening tests and the lack of direct relationship between the titers, thermal threshold and risk of complications makes the screening an uncommon practice.

Ruptured pulmonary artery aneurysm: a surgical emergency.

Idiopathic pulmonary artery aneurysm rupture was diagnosed in a 79-year-old man who presented with a dry cough. He was considered unlikely to tolerate extensive pulmonary artery reconstruction or lung resection; hence, he was salvaged by timely ligation of the distal pulmonary artery at the origin of the aneurysm.

Role of the inflammasome, IL-1ß, and IL-18 in bacterial infections.

The inflammasome is an important innate immune pathway that regulates at least two host responses protective against infections: (1) secretion of the proinflammatory cytokines IL-1ß and IL-18 and (2) induction of pyroptosis, a form of cell death. Inflammasomes, of which different types have been identified, are multiprotein complexes containing pattern recognition receptors belonging to the Nod-like receptor family or the PYHIN family and the protease caspase-1. The molecular aspects involved in the activation of different inflammasomes by various pathogens are being rapidly elucidated, and their role during infections is being characterized. Production of IL-1ß and IL-18 and induction of pyroptosis of the infected cell have been shown to be protective against many infectious agents. Here, we review the recent literature concerning inflammasome activation in the context of bacterial infections and identify important questions to be answered in the future.

Channel catfish CD8α and CD8ß co-receptors: characterization, expression and polymorphism.

In this study we report the identification and characterization of channel catfish, Ictalurus punctatus CD8α and CD8ß genes. Both genes encode predicted proteins containing a leader, a immunoglobulin superfamily V domain, a stalk/hinge region, a transmembrane region and a positively charged cytoplasmic tail (CYT) containing the conserved teleost C-X-H motif. Catfish CD8α and CD8ß are encoded as single copy genes and as in other vertebrates exhibit a conserved head to tail synteny; the CD8ß gene is found 14.1kb upstream of the CD8α gene. Both CD8α and CD8ß transcripts showed a low degree of polymorphism. Finally, as determined by q-PCR both CD8α and CD8ß are expressed in various catfish lymphoid tissues with the highest expression observed in thymus from 2 month old catfish-fry. In the future these results will provide the basis for evaluating the role of CD8(+) CTL and other CD8-bearing cells in response to immunization or infection in the catfish.

Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1ß is deleterious.

Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1ß, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1ß and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1ß by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3⁻/⁻ mice was due to decreased production of IL-18 and IL-1ß. In contrast, Nlrc4⁻/⁻ mice produced IL-1ß and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1ß or IL-1 receptor agonist revealed that IL-1ß has deleterious effects during melioidosis. The detrimental role of IL-1ß appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4⁻/⁻ mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1ß.

Identification of two IgD+ B cell populations in channel catfish, Ictalurus punctatus.

Channel catfish Ictalurus punctatus express two Ig isotypes: IgM and IgD. Although catfish IgM has been extensively studied at the functional and structural levels, much less is known about IgD. In this study, IgM(+)/IgD(+) and IgM(-)/IgD(+) catfish B cell populations were identified through the use of anti-IgM and anti-IgD mAbs. Catfish IgM(+)/IgD(+) B cells are small and agranular. In contrast, IgM(-)/IgD(+) B cells are larger and exhibit a plasmablast morphology. The use of cell sorting, flow cytometry, and RT-PCR demonstrated that IgD(+) B cell expression varies among individuals. For example, some catfish have <5% IgM(-)/IgD(+) B cells in their PBLs, whereas in others the IgM(-)/IgD(+) B cell population can represent as much as 72%. Furthermore, IgD expressed by IgM(-)/IgD(+) B cells preferentially associates with IgL σ. Comparatively, IgM(+)/IgD(+) B cells can express any of the four catfish IgL isotypes. Also, transfection studies show that IgD functions as a typical BCR, because Igδ-chains associate with CD79a and CD79b molecules, and all membrane IgD transcripts from sorted IgM(-)/IgD(+) B cells contain viable VDJ rearrangements, with no bias in family member usage. Interestingly, all secreted IgD transcripts from IgM(+)/IgD(+) and IgM(-)/IgD(+) B cells were V-less and began with a leader spliced to Cδ1. Importantly, transfection of catfish clonal B cells demonstrated that this leader mediated IgD secretion. Together, these findings imply that catfish IgM(-)/IgD(+) B cells likely expand in response to certain pathogens and that the catfish IgD Fc-region, as has been suggested for human IgD, may function as a pattern recognition molecule.

Characterization of anti-channel catfish IgL sigma monoclonal antibodies.

This study characterizes three monoclonal antibodies (mAbs) developed against the constant (C) region of the immunoglobulin light (IgL) sigma chain isotype of the channel catfish, Ictalurus punctatus. Microsphere bead assays and Western blot analyses utilizing different recombinant (r) proteins show that these anti-catfish IgL sigma chain mAbs each specifically recognize the denatured form of IgL sigma. Importantly, Western blotting of catfish sera using the anti-IgL sigma mAbs also identified an IgL chain-sized immunoreactive band(s) of approximately 27kDa. It is anticipated that these mAbs in combination with the already existing anti-catfish Ig heavy (H) and IgL chain mAbs will be useful in future studies examining the functional roles of the different catfish IgL isotypes.

Identification of Igsigma and Iglambda in channel catfish, Ictalurus punctatus, and Iglambda in Atlantic cod, Gadus morhua.

Immunoglobulin light (IGL) chain genes encoding sigma and lambda from channel catfish, Ictalurus punctatus, and lambda from Atlantic cod, Gadus morhua, were identified by mining of expressed sequence tag databases, 5'-RACE and RT-PCR protocols. cDNAs for each of these IGL chains encode typical variable (V), joining (J), and constant (C) regions and Southern blot analyses and genomic sequencing show that genes encoding these isotypes, like other teleost IGL genes, are found in a cluster organization of one or two V gene segments, followed by single J and C gene segments, all in the same transcriptional orientation. However, unlike the teleost kappa genes, genes encoding catfish sigma and lambda are few in number and the two isotypes are each encoded by only two clusters. Similarly, Atlantic cod lambda genes are predicted to be encoded by two or three clusters. As expected, sequence and phylogenetic analyses comparisons demonstrate that catfish Vsigma and Csigma genes are most similar to Vsigma and Csigma genes of other ectothermic vertebrates. Although catfish and Atlantic cod Vlambda genes cluster with other vertebrate Vlambda genes, their Clambda sequences cluster in a distinct group separate from other vertebrate IGL C sequences. However, support for classifying these sequences as lambda, is their V and J recombination signal sequence (RSS) organization. The catfish and Atlantic cod genes have typical lambda-like RSS with the Vlambda RSS consisting of heptamer-23 bp spacer-nonamer and the Jlambda RSS consisting of heptamer-12 bp spacer-nonamer. This is the first report demonstrating the presence of Iglambda in teleosts.

Early anticoagulation after mechanical valve implantation, and related complications.

BACKGROUND AND AIM OF THE STUDY: Anticoagulation is started soon after mechanical valve replacement as the risk of thromboembolic complications is especially high during the first six months after surgery. At present there is no consensus on the optimal protocol to prevent early thrombogenic complications, without increasing the risk of postoperative hemorrhagic events. Herein is presented a comparative analysis of the various anticoagulation protocols utilized at the authors' institution. METHODS: Between July 2001 and October 2006, a total of 503 patients underwent mechanical valve implantation at the authors' institution. The patients were allocated to three comparable groups, depending on the anticoagulation regime administered. Group A patients (n = 221) received only oral anticoagulation from the first postoperative day; group B patients (n = 159) received oral anticoagulation plus low-molecular weight heparin; and group C patients (n = 123) received unfractionated heparin within 12 h of surgery in addition to oral anticoagulation. RESULTS: At 48 h after surgery the mean postoperative drainage was 514.1 +/- 202 ml, 783.4 +/- 369.7 ml, and 718.4 +/- 305.5 ml in groups A, B and C, respectively. Two patients in group A, 12 in group B and nine in group C required the reinsertion of additional intercostal/pericardial drains for collections (p = 0.002). Twelve patients had tamponade (seven in group B, five in group C; p = 0.002), and nine (five in group B, four in group C) required re-exploration for excessive drainage at >48 h after surgery (p = 0.01). There were three incidents of valve thrombosis within the first postoperative six months (one in each group). Two of these patients had a suboptimal International Normalized Ratio (INR), while the third patient had an INR >5 with congestive heart failure with hepatic failure. All three were successfully thrombolyzed and recovered after initial ventilatory and inotropic support. The incidence of thromboembolic stroke was low in all groups. CONCLUSION: Early oral anticoagulation alone provides optimum anticoagulation and is associated with minimum complications. Early supplementation with heparin increases the risk of hemorrhagic complications but without reducing the thromboembolic risk.

B cell receptor accessory molecules in the channel catfish, Ictalurus punctatus.

B cell receptor (BCR) accessory molecules CD79a and CD79b homologs were identified in the channel catfish, Ictalurus punctatus. Both are found as single copy genes that encode proteins containing a signal peptide, an extracellular immunoglobulin domain, a transmembrane region and a cytoplasmic tail containing an immune-receptor tyrosine-dased activation motif (ITAM). IpCD79a and IpCD79b transcripts correlate well with IgM message expression. They are highly expressed in peripheral blood leukocytes (PBL) enriched in membrane (m) IgM+ cells and catfish clonal B cell lines, but not in catfish clonal T cells, indicating that IpCD79a and IpCD79b expression is B cell restricted. Studies using catfish clonal B cells (3B11) transfected with constructs encoding epitope-tagged IpCD79a and IpCD79b revealed that IpCD79a was expressed as a 45 kDa protein and IpCD79b was expressed as a 32 kDa protein. Furthermore, co-immunoprecipitations of epitope-tagged CD79 proteins demonstrate that these molecules are non-covalently associated with mIgM. These data correlate with some of the previous immunoprecipitation data demonstrating that catfish mIgM associates with proteins of 45 and 32 kDa.