Regulatory T cells in spontaneous autoimmune encephalomyelitis

Summary: Spontaneous experimental autoimmune encephalomyelitis (EAE) develops in 100% of mice harboring a monoclonal myelin basic protein (MBP)-specific CD4⁺αβ T-cell repertoire. Monoclonality of the αβ T-cell repertoire can be achieved by crossing MBP-specific T-cell receptor (TCR) transgenic mice with either RAG^−/− mice or TCR α^−/−/TCR β^−/− double knockout mice. Spontaneous EAE can be prevented by a single administration of purified CD4⁺ splenocytes or thymocytes obtained from wild-type syngeneic mice. The regulatory T cells (T-reg) that protect from spontaneous EAE need not express the CD25 marker, as effective protection can be attained with populations depleted of CD25⁺ T cells. Although the specificity of the regulatory T cells is important for their generation or regulatory function, T cells that protect from spontaneous EAE can have a diverse TCR α and β chain composition. T-reg cells expand poorly in vivo , and appear to be long lived. Finally, precursors for T-reg are present in fetal liver as well as in the bone marrow of aging mice. We propose that protection of healthy individuals from autoimmune diseases involves several layers of regulation, which consist of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁻ T-reg cells, and anti-TCR T cells, with each layer potentially operating at different stages of T-helper cell-mediated immune responses.     

Insulin hypersensitivity in mice lacking the V1b vasopressin receptor

We have reported that [Arg⁸]-vasopressin-stimulated insulin release is blunted in islet cells isolated from V1b receptor-deficient ( V1bR ^−/−) mice. In this study, we used V1bR ^−/− mice to examine the physiological role of the V1b receptor in regulating blood glucose levels in vivo , and we found that the fasting plasma glucose, insulin and glucagon levels were lower in V1bR ^−/− mice than in wild-type ( V1bR ^+/+) mice. Next, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose and insulin levels during the GTT were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. An insulin tolerance test (ITT) revealed that, after insulin administration, plasma glucose levels were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. In addition, a hyperinsulinaemic–euglycaemic clamp study showed that the glucose infusion rate was increased in V1bR ^−/− mice, indicating that insulin sensitivity was enhanced at the in vivo level in V1bR ^−/− mice. Furthermore, we found that the V1b receptor was expressed in white adipose tissue and that insulin-stimulated phosphorylation of Akt as an important signaling molecule was increased in adipocytes isolated from V1bR ^−/− mice. Thus, the blockade of the V1b receptor could result, at least in part, in enhanced insulin sensitivity by altering insulin signalling in adipocytes.     

Major Histocompatibility Complex Class II Region Confers Susceptibility to Staphylococcus aureus Arthritis

The importance of the MHC class II region for the development of septic arthritis was studied in a murine model of haematogenously induced Staphylococcus aureus arthritis. In the first experiment MHC class II deficient mice (Aβ^−/−) and their heterozygous (Aβ^+/−) littermates were intravenously inoculated with a single dose of toxic shock syndrome toxin-1 producing S. aureus LS-1 strain. The results demonstrate that the expression of class II MHC molecules increases the prevalence and severity of arthritis. To analyse the impact of MHC class II haplotypes on the disease onset and progression the authors used congenic C3H.NB, C3H.Q and C3H/HeJ mice in the second set of experiments. The results show that C3H/HeJ mice developed the highest frequency and the most severe course of arthritis compared with C3H.NB and C3H.Q animals. Immunohistochemical analysis of arthritic joints revealed equal number of macrophages, CD4⁺ and CD8⁺ lymphocytes in the inflamed synovia in all the congenic mice. In contrast, the number of MHC class II expressing cells was higher in the arthritic joints of C3H/HeJ mice compared with the congenic strains ( P <0.001). Furthermore, serum levels of proarthrtitogenic cytokines, such as tumour necrosis factor and interleukin-6 were higher in C3H/HeJ group. This study indicates that MHC class II expression is necessary for the development of S. aureus arthritis in mice and that different MHC class II haplotypes confer varying susceptibility for development of joint inflammation induced by staphylococci.     

γδ T cells: firefighters or fire boosters in the front lines of inflammatory responses

Summary:  Intradermal inoculation of cloned self-reactive αβ T cells into the footpads of mice induced cutaneous graft-versus-host disease (GVHD), and after recovery from GVHD, the epidermis became resistant to subsequent attempts to induce GVHD. Resistance to GVHD was not induced in the epidermis of T-cell receptor δ-deficient (TCRδ^−/−) mice that lacked γδ T cells bearing canonical Vγ5/Vδ1⁺γδTCRs, known as dendritic epidermal T cells (DETCs), and resistance was restored by reconstitution of these mutant mice with precursors of Vγ5⁺ DETCs. Pulmonary infection by Cryptococcus neoformans induced an increase of γδ T cells in the lung, and in comparison with wildtype mice, TCRδ^−/− mice eliminated C. neoformans more rapidly and synthesized more interferon-γ in the lung. In the mouse small intestine, the absence of γδ T cells is associated with a reduction in epithelial cell turnover and downregulation of the expression of major histocompatibility complex class II molecules. The protective role of γδ T cells was verified in a dextran sodium sulfate-induced inflammatory bowel disease (IBD) model, whereas in a spontaneous model of IBD, γδ T cells were involved in the exacerbation of colitis in TCRα^−/− mice. Taken together, in addition to the homeostatic regulation of epithelial tissues, γδ T cells appear to play a pivotal role in the modification of inflammatory responses induced in many organs containing epithelia.     

CD3−4−8− Thymocyte Precursors with Interleukin-2 Receptors Differentiate Phenotypically in Coculture with Thymic Stromal Cells

CD3⁻4⁻8⁻ interleukin-2 receptor positive (IL-2R⁺) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R⁺CD3⁻4⁻8⁻ thymocytes were obtained by positive panning of IL-2R⁺ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2½ days significant numbers of CD4⁺8⁺ and CD3⁺4⁺8⁻ cell populations developed, the latter expressing the αβ T-cell receptor (αβ-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R⁺ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.     

Immunological Response of Major Histocompatibility Complex Class II-Deficient (Aβ°) Mice Infected by the Parasite Schistosoma mansoni

We have characterized the immunological behaviour of major histocompitibility complex (MHC) Class II molecule-deficient (Aβ°) mice after infection by Schistosoma mansoni . In Aβ° mice, morbidity developed dramatically 7 weeks after infection leading to death, despite the absence of an increase in parasite burden or of eggs trapped in the liver. Histological examination of the liver revealed the absence of a classical granulomatous reaction. Antibodies were produced only against schistosomulum antigens. Specific antibodies against adult worm (SWAP) or egg antigen (SEA) were not detected. Cytokine production (IFN-γ and IL-4) was absent after in vitro restimulation of splenic cells from infected Aβ° mice with parasite antigens. Adoptive transfer of primed splenic cells (total, purified CD4⁺ or CD8⁺ T cells) failed to improve survival or to induce a granulomatous reaction in infected Aβ° mice. Survival, cellular and humoral responses in CD8⁺ T-cell-depleted Aβ° mice or MHC° mice (lacking MHC class I and II molecules) were similar to nondepleted Aβ° mice, suggesting that anti-schistosomula antibody production was thymo-independent. Our results demonstrate a high degree of susceptibility of Aβ° mice to infection and corroborate the importance of CD4⁺ T cells in the initiation of the granulomatous response. However, our results do not show evidence for the involvement of CD8⁺ T cells in response to S. mansoni infection.     

Glycinergic input of widefield, displaced amacrine cells of the mouse retina

Glycine receptors (GlyRs) of displaced amacrine cells of the mouse retina were analysed using whole cell recordings and immunocytochemical staining with subunit-specific antibodies. During the recordings the cells were filled with a fluorescent tracer and 11 different morphological types could be identified. The studies were performed in wild-type mice and in mutant mice deficient in the GlyRα1 ( Glra1^spd-ot , 'oscillator' mouse), the GlyRα2 ( Glra2 ^−/−) and the GlyRα3 subunit ( Glra3 ^−/−). Based on their responses to the application of exogenous glycine in the retinas of wild-type and mutant mice, the cells were grouped into three major classes: group I cells (comprising the morphological types MA-S5, MA-S1, MA-S1/S5, A17, PA-S1, PA-S5 and WA-S1), group II cells (comprising the morphological types PA-S4, WA-S3 and WA-multi) and ON-starburst cells. For further analysis, spontaneous inhibitory postsynaptic currents (sIPSCs) were measured both in wild-type and mutant mouse retinas. Glycinergic sIPSCs and glycine induced currents of group I cells remained unaltered across wild-type and the three mutant mice (mean decay time constant of sIPSCs, τ∼25 ms). Group II cells showed glycinergic sIPSCs and glycine induced currents in wild-type, Glra1^spd-ot and Glra3 ^−/− mice (τ∼25 ms); however, glycinergic currents were absent in group II cells of Glra2 ^−/− mice. Glycine induced currents and sIPSCs recorded from ON-starburst amacrine cells did not differ significantly between wild-type and the mutant mouse retinas (τ∼50–70 ms). We propose that GlyRs of group II cells are dominated by the α2 subunit; GlyRs of ON-starburst amacrine cells appear to be dominated by the α4 subunit.     

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion

Parietal cell (PC) proton secretion via H⁺/K⁺-ATPase requires apical K⁺ recycling. A variety of K⁺ channels and transporters are expressed in the PC and the molecular nature of the apical K⁺ recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7- to 8-day-old KCNQ1^+/+, ^+/– and ^−/− mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1^−/− and ^+/+ mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin-stimulated acid output reduced by ∼90% in KCNQ1^−/− gastric mucosa. Application of a high K⁺ concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1^−/− mucosa. The study demonstrates that the KCNQ1 channel provides K⁺ to the extracellular K⁺ binding site of the H⁺/K⁺-ATPase during acid secretion, and no other gastric K⁺ channel can substitute for this function.     

Tumor necrosis factor alpha is reparative via TNFR2 [corrected] in the hippocampus and via TNFR1 [corrected] in the striatum after virus-induced encephalitis

Differentiating between injurious and reparative factors facilitates appropriate therapeutic intervention. We evaluated the role of tumor necrosis factor α (TNFα) in parenchymal brain pathology resolution following virus-induced encephalitis from a picornavirus, Theiler's murine encephalomyelitis virus (TMEV). We infected the following animals with TMEV for 7 to 270 days: B6/129 TNF^−/− mice (without TNFα expression), B6/129 TNFR1^−/− mice (without TNFα receptor 1 expression), and B6/129 TNFR2^−/− mice (without TNFα receptor 2 expression). Normal TNFα-expressing controls were TMEV-infected B6, 129/J, B6/129F1 and B6/129F2 mice. Whereas all strains developed inflammation and neuronal injury in the hippocampus and striatum 7 to 21 days postinfection (dpi), the control mice resolved the pathology by 45 to 90 dpi. However, parenchymal hippocampal and striatal injury persisted in B6/129 TNF^−/− mice following infection. Treating virus-infected mice with active recombinant mouse TNFα resulted in less hippocampal and striatal pathology, whereas TNFα-neutralizing treatment worsened pathology. T1 "black holes" appeared on MRI during early infection in the hippocampus and striatum in all mice but persisted only in TNF^−/− mice. TNFR1 mediated hippocampal pathology resolution whereas TNFR2 mediated striatal healing. These findings indicate the role of TNFα in resolution of sublethal hippocampal and striatal injury.     

Impaired Antibody Responses but Normal Proliferation of Specific CD4+ T Cells in Mice Lacking Complement Receptors 1 and 2

Severely impaired Ab responses are seen in animals lacking C (complement) factors C2, C3 or C4 as well as CR1/2 (C receptors 1 and 2). The molecular mechanism behind this phenomenon is not understood. One possibility is that C-containing immune complexes are endocytosed via CR2 on B cells and presented to specific CD4⁺ T cells, which would then proliferate and provide efficient help to specific B cells. In vitro , B cells can endocytose immune complexes via CR1/2 and present the Ag to T cells. Whether absence of this Ag presenting function in Cr2^−/− mice (mice lacking CR1/2) explains their low Ab response is unclear. To address this question, Cr2^−/− and wild type mice were transferred with OVA-specific T cells, obtained from the DO11.10 strain which has a transgenic TCR recognizing an OVA peptide. The animals were subsequently immunized with sheep red blood cells (SRBC) conjugated to OVA. Interestingly, proliferation of the OVA-specific T cells was normal in Cr2^−/− mice, although their Ab response to both SRBC and OVA was severely impaired. These observations suggest that the impaired Ab response in Cr2^−/− mice cannot be explained by a lack of appropriate induction of T cell help.     

Population Dynamics of CD4+ T Cells Lacking Thy-1 in Murine Retrovirus-Induced Immunodeficiency Syndrome (MAIDS)

Increased numbers of CD4⁺ Thy-1 cells have been described in the spleen (SP) of mice with retrovirusinduced immunodeliciency (MAIDS). Since this phenotypic abnormality might have considerable functional importance, the expansion of the CD4⁺ Thy-1 subset in MAIDS was characterized further. CD4⁺ Thy-1⁻ and Thy-1⁺ T-cell is from infected mice expressed similar densities of CD3 and TCR γ/β. In contrast, the Thy-I⁻ subset was uniformly CD44^hi, even early in the disease when part of Thy-I⁺ cells were still CD44¹⁰. The emergence of CD4⁺ Thy-1⁻cells occurred first in SP and lymph nodes and was observed later in thymus. The important fraction ofCD4⁺ cells lacking Thy-1 normally present in Peyer's patches was only weakly modified. Despite the major expansion of the CD4⁺ Thy-1⁻ phenotype. the proliferating fraction was not higher in this subset than in CD4⁺ Thy-1⁺ cells from infeeted miee. Persistence after hydroxyurea administration was identical in both subsets, indicating similar mean cell lifespans. Taken together, these results show that the major expansion of CD4⁺ Thy-I⁻ T-cells in MAIDS is not ascribable solely to increased proliferation within this subset. Phenotypic analysis suggests that CD4⁺ Thy-I⁻ cells result from the differentiation of Thy-I⁺ cells induced by activation signals related to retroviral infection.     

Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.     

Peripheral Lymphoid Hyperplasia and Central Lymphoid Depletion in Mice Treated with a Bacterial B-Cell Mitogen (F3'EP-Si/p90)

In order to further understand the mechanism mediating the mitogenic and immunosuppressor effects of p90, a protein produced by Streptococcus intermedius , flow cytometric studies were performed on peripheral and central lymphoid organs of mice treated with this protein. p90 induced a strong blastogenic B-cell response in the spleen and lymph nodes, followed by a slight but significant polyclonal T-cell activation. B-cell repertoire analysis indicated that polyclonal B-cell responses affected similarly both CD5⁺ and conventional (CD5⁻) B cells in the spleen. Repertoire analysis of T cells failed to reveal any preferential stimulation of the Vβ T-cell receptor (Vβ-TcR) families studied. Peripheral lymphoid hyperplasia was observed concomitantly with central lymphoid depiction. In the bone marrow, pre-B and B cells were profoundly depleted, with a more pronounced effect on small pre-B cells. In the thymus, double-positive (CD4⁺ CD8⁺) thymocytes were preferentially eliminated, with a relative enrichment of single positive (either CD4⁺ or CD8⁺) and double-negative (CD4⁻CD8⁻) thymocytes.     

II. The Effects of Thalidomide Treatment on Autoimmune-Prone NZB and MRL Mice are Consistent with Stimulation of the Central Immune System

We describe here some immunomodulatory effects of thalidomide on autoimmune-prone mice. The highly increased synthesis of splenic IgM in NZB mice, of splenic and lymph node IgG of different subclasses in MRL/n mice, and of splenic and lymph node IgGl in MRL/lpr mice was markedly inhibited by thalidomide treatment. After a single treatment with 3mg of thalidomide, the following changes were observed in NZB mice: (i) an initial decrease in the numbers of large CD5⁺μ^high, and in the numbers of total CD5⁺μ⁻, CD5⁻μ^high, CD5⁺μ^high lymphocyte populations of the pleural cavity followed by a late increase in the numbers of large cells of the three cell populations; (ii) a consistent increase in the numbers of a CD5^lowμ^low pleural lymphoid population; (iii) a consistent reduction in the numbers of splenic large CD5⁺ B cells and an oscillatory increase in the number of cells with CD5⁻ phenotype; (iv) a late reduction in the numbers of splenic total CD5⁺ B cells. These results are consistent with the notion that thalidomide controls a disease-associated expansion of B cells in autoimmune prone mouse strains through a stimulatory effect of the drug on the immune system.     

IL-12p40 is essential for the down-regulation of airway hyperresponsiveness in a mouse model of bronchial asthma with prolonged antigen exposure

Background We previously reported a mouse model of bronchial asthma showing eosinophilic inflammation, but not airway hyperresponsiveness (AHR), after prolonged antigen exposure. This model showed an increase of IL-12 in the lung.
Objective The aim of this study was to investigate the role of IL-12p40 in a murine asthma model with prolonged antigen exposures.
Methods An ovalbumin (OVA)-induced asthma model was first established in wild-type (WT) and IL-12p40-deficient (IL-12p40^−/−) mice. Both strains of mice were further exposed to either OVA (prolonged exposure group) or phosphate-buffered saline (positive control group) 3 days per week for 3 weeks. During week 4, both groups of mice were given a final challenge with OVA.
Results Prolonged antigen exposures resulted in marked suppression of airway eosinophilia in both WT and IL-12p40^−/− mice. However, AHR persisted in IL-12p40^−/− but not in WT mice. There were no significant differences of IL-5, IL-13 or IFN-γ levels in bronchoalveolar lavage fluid between WT and IL-12p40^−/− mice. The hydroxyproline content of the lung and peribronchial fibrosis were, however, significantly increased in IL-12p40^−/− mice.
Conclusion The results suggest that endogenous IL-12p40 is essential for inhibition of AHR and peribronchial fibrosis, but not eosinophilic inflammation, in a murine asthma model with prolonged antigen exposures.     

Congenital tracheal malformation in cystic fibrosis transmembrane conductance regulator-deficient mice

In cystic fibrosis (CF) patients, the major alteration in pulmonary function is due to peripheral airway obstruction. In the present study, we investigated the possibility that alterations in the extrathoracic airways, particularly in the trachea that expresses high levels of CFTR (CF transmembrane conductance regulator), may contribute to respiratory dysfunction. We performed morphological analyses of the trachea and airway functional studies in adult Cftr knockout ( Cftr ^−/−) and F508del-CFTR mice and their controls. Macroscopic and histological examination of the trachea showed the presence of one to seven disrupted or incomplete cartilage rings in Cftr ^−/− mice (23/25) while only a few Cftr^+/+ mice (6/25) had one abnormal ring. Tracheal defects were mainly localized in the proximal trachea. In 14 Cftr ^−/− mice, frontal disruption of the first three to six rings below the cricoid cartilage were associated with upper tracheal constriction. Similar tracheal abnormalities were detected in adult F508del-CFTR and in newborn Cftr^−/− and F508del-CFTR mice. Tracheal and ventilatory function analyses showed in Cftr ^−/− mice a decreased contractile response of the proximal trachea and a reduced breathing rate due to an increase in the inspiratory and expiratory times. In F508del-CFTR mice, the expiratory time was longer than in controls. Therefore, these structural and functional abnormalities detected in adult and newborn CF mouse models may represent congenital malformations related to CFTR dysfunction. These results raise important questions concerning the mechanisms governing tracheal development within the context of CFTR protein dysfunction and the implication of such abnormalities in the pathogenesis of airway disease in CF.     

Tumor Necrosis Factor-α-Associated Mechanisms Affecting the Embryonic Response to Cyclophosphamide

Problem  We have previously shown that TNF-α^−/− embryos are more sensitive to the exposure to cyclophosphamide (CP) compared with TNF-α^+/+ embryos; however, the underlying mechanisms are not fully understood. Thus, in our present study, we tried to identify those molecules that might be responsible for the protective effect of the cytokine.
Method of study  CP-treated TNF-α^−/− and TNF-α^+/+ embryos were analyzed for changes in apoptosis by TUNEL and flow cytometry, while cell proliferation was analyzed by BrdU incorporation. The expression of Bax, bcl-2, p53, the p65 subunit of NF-κB and IκBα was assessed by Western blotting and immunohistochemistry.
Results  CP-treated TNF-α^−/− embryos exhibited a more profound decrease in their weight, which was accompanied by an earlier appearance of cellular damage and apoptotic cells and an earlier decrease in cell proliferation in the embryonic brain compared with TNF-α^+/+ embryos. Also, an increased percentage of Bax-positive cells and a decreased percentage of bcl-2-positive cells were detected in TNF-α^−/− embryos 48 hr after exposure, which were accompanied by a decreased percentage of p53-positive cells.
Conclusion  Our data implicate TNF-α to be involved in the protection of the embryo against CP teratogenicity, possibly via alteration in Bax, bcl-2 or p53 expression.     

Peripheral T-cell Lymphomas Immunologic and Enzyme Histochemical Analysis of 15 Cases

Ffteen cases of peripheral T cell lymphoma were studied to evaluate the respective properties of various histologic types using enzyme histochemical and ultrastructural examinations in addition to immunological methods. Eleven cases in an ATLA negative group manifested various histologic patterns such as IBL like, pleomorphic and Lennert's lymphomas in comparison with the relatively monomorphic proliferation of neoplastic lymphoid cells in the 4 ATLA positive cases. The presence of neoplastic clear cells is characteristic of peripheral T-cell malignancies, and is likely to be found in CD4 lymphomas. There is an occasional reaction of epithelioid histiocytes and plasma cells with eosinophils, the former being designated Lennert's lymphoma and the latter IBL like T-cell lymphoma. Immunological examination revealed four immunophenotypic patterns: (1) CD2⁺3⁺4⁺8⁺, (2) CD2⁺ 3⁻4⁺8⁻, (3) CD2⁺3⁺4⁻8⁺, and (4) CD2⁺3⁺4⁺8⁺, but did not provide information concerning the intimate relationship between histologic types and immuno phenotyes. β-Glucuronidase reactivity, however, contributed to the distinction between helper and suppressor T cell malignancies, suggesting its usefulness for distinguishing these two cell types and their malignant counterparts.     

Carbonic anhydrase gene expression in CA II-deficient (Car2−/−) and CA IX-deficient (Car9−/−) mice

Using real-time PCR and immunohistochemistry, we have examined the expression of carbonic anhydrase isozymes (CA) I, II, III, IV, IX, XII, XIII and XIV in the brain, kidney, stomach and colon of the wild-type, CA II-deficient ( Car2^−/− ), and CA IX deficient ( Car9^−/− ) mice. The expression of Car4, Car12, Car13 and Car14 mRNAs did not show any significant deviations between the three groups of mice, whereas both groups of CA deficient mice showed decreased expression levels of Car1 in the colon and Car3 in the kidney. The Car2 mRNA level was greatly reduced but not completely abolished in all four tissues from the Car2^−/− mice in which no CA II protein was expressed. Sequencing the Car2 cDNA isolated from C57BL6 Car2^−/− mice revealed two nucleotide differences from the wild-type C57BL6 mice. One is a silent polymorphism found in Car2 mRNA from wild-type DBA mice, which is the strain that provided the original mutagenized chromosome. The second change is a mutation that causes prematurely terminated translation at codon 155 (Gln155X). Car9 mRNA and CA IX protein expression levels were up-regulated about 2.5- and 3.6-fold, respectively, in the stomach of the Car2^−/− mice. These results suggest that the loss of function of cytosolic CA II in the stomach of Car2^−/− mice leads to up-regulation of an extracellular CA, namely CA IX, which is expressed on the cell surface of the gastric epithelium.