Image analyzer-based assessment of tumor-infiltrating T cell subsets and their prognostic values in colorectal carcinomas.

To find useful tools to evaluate the prognosis in colorectal carcinoma (CRC) patients, we investigated the prognostic values of tumor-infiltrating T lymphocyte subsets according to intratumoral subsites as well as clinical or molecular characteristics. Immunohistochemistry for CD8, CD45RO, and FOXP3 was performed, and densities of the T cell subsets in each tissue microarray core (cells/mm2) were measured by image analysis. In the training set (n = 218) of CRC, T cell subset densities in the invasion front were more strongly associated with patient outcome than those in the tumor center. In the validation set (n = 549), T cell subset densities in the invasion front were evaluated. Univariate analysis showed that all three T cell subset densities were significantly associated with longer progression free survival and overall survival time (p < 0.001). In multivariate analysis, a high CD45RO density correlated independently with longer progression free survival (p = 0.011) and overall survival time (p = 0.007) in CRC patients, regardless of tumor location or adjuvant chemotherapy status. Our results showed that CD45RO density in the invasion front was the only independent prognostic factor regarding CRC. However, CD8 and FOXP3 densities were also independent prognostic factors in certain clinical settings. Thus, image analysis of tissue microarray cores in the invasion front of CRC could be used as a valid method for evaluating the prognostic significance of T cell subset densities.

Immunomagnetic CD45 depletion does not improve cytokeratin 20 RT-PCR in colorectal cancer.

BACKGROUND: Cytokeratin 20 reverse transcriptase polymerase chain reaction (CK20 RT-PCR) of blood and bone marrow specimens has been suggested for assessment of hematogenously disseminated tumor cell (DTC) spread in colorectal cancer (CRC) patients. Considerable discrepancies among the studies reported indicate a need for better evaluation procedures. We investigated whether mononucleated cell (MNC) enrichment by Ficoll density gradient centrifugation followed by immunomagnetic depletion of CD45-positive cells (extended enrichment) allows better detection of DTC-associated CK20 mRNA compared to MNC enrichment by Ficoll density gradient centrifugation alone (Ficoll enrichment). METHODS: We analyzed 53 samples [38 peripheral blood (PB), 15 bone marrow (BM)] from 38 CRC patients. Extended enrichment was performed for 30 specimens (PB and BM, n=15 each), and Ficoll enrichment for 23 blood specimens. Total RNA was extracted, reverse-transcribed and analyzed by real-time RT-PCR using a LightCycler instrument. RESULTS: Despite extended enrichment, 10 PB and 8 BM samples could not be analyzed because of low cellular yield. The depletion efficiency of CD45 separation was 2 log. RT-PCR of the housekeeping gene PBGD resulted in high and varied crossing point values (mean 37.1+3.0) for five PB and seven BM specimens. Ficoll enrichment yielded 23 analyzable blood specimens for which the mean crossing point value was 26.7+0.5 in PBGD RT-PCR. CK20 RT-PCR of 23 blood samples (all from Dukes D patients) revealed CK20 transcripts in four cases (17%). CONCLUSIONS: Extended enrichment was not superior to Ficoll enrichment; in fact, the sensitivity was lower. Improvement of the reported CK20 RT-PCR assay of Ficoll-enriched MNC populations is warranted.

Profiling of the CD4 receptor complex proteins.

Intermolecular complexes produced by the CD4 molecule were studied. To preserve the integrity of weak protein-protein interactions of the CD4 antigen, cells were lysed in a mild nonionic detergent Brij97. Protein constituents of the complex were identified by our previously proposed fluorescence immunoprecipitation assay with subsequent mass spectrometry. In total, 26 proteins associated with CD4 were identified on CEM cells. The CD4 complex included the following major components: tyrosine phosphatase CD45, transferrin receptor CD71, tyrosine kinase Lck, and a lymphocyte phosphatase-associated phosphoprotein LPAP. The CD4 complex also contained some components of cytoskeleton and heat shock proteins. The association between CD4, CD71, and CD45 molecules was confirmed by immunoblotting. The CD4 complexes were not detected on the U937 myeloid cells lacking Lck and LPAP. We attempted to quantitatively characterize the CD4 complex composition.

Isolation and characterization of nontubular sca-1+lin- multipotent stem/progenitor cells from adult mouse kidney.

Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Recently, stage-specific embryonic kidney progenitor tissue was used to regenerate nephrons. Through fluorescence-activated cell sorting, microarray analysis, in vitro differentiation assays, mixed lymphocyte reaction, and a model of ischemic kidney injury, this study sought to identify and characterize multipotent organ stem/progenitor cells in the adult kidney. Herein is reported the existence of nontubular cells that express stem cell antigen-1 (Sca-1). This population of small cells includes a CD45-negative fraction that lacks hematopoietic stem cell and lineage markers and resides in the renal interstitial space. In addition, these cells are enriched for beta1-integrin, are cytokeratin negative, and show minimal expression of surface markers that typically are found on bone marrow-derived mesenchymal stem cells. Global gene profiling reveals enrichment for many genes downstream of developmental signaling molecules and self-renewal pathways, such as TGF-beta/bone morphogenic protein, Wnt, or fibroblast growth factor, as well as for those that are involved in specification of mesodermal lineages (myocyte enhancer factor 2A, YY1-associated factor 2, and filamin-beta). In vitro, they are plastic adherent and slowly proliferating and result in inhibition of alloreactive CD8(+) T cells, indicative of an immune-privileged behavior. Furthermore, clonal-derived lines can be differentiated into myogenic, osteogenic, adipogenic, and neural lineages. Finally, when injected directly into the renal parenchyma, shortly after ischemic/reperfusion injury, renal Sca-1(+)Lin(-) cells, derived from ROSA26 reporter mice, adopt a tubular phenotype and potentially could contribute to kidney repair. These data define a unique phenotype for adult kidney-derived cells, which have potential as stem cells and may contribute to the regeneration of injured kidneys.

Carcinoma de células pequeñas de glándula salival submaxilar / Small cell carcinoma of the submaxillary gland

El carcinoma de células pequeñas de la glándula salivalsubmaxilar es una entidad rara en esta localización. Para sudiagnóstico definitivo es importante realizar estudio inmunohistoquímicoy descartar la posibilidad de metástasis. Eneste artículo presentamos un nuevo caso en un paciente de76 años, sin patología pulmonar conocida y revisamos laliteratura publicada hasta la fecha

Background: Small cell carcinoma in the submaxillarygland is a very rare entity. To achieve a correct pathologicaldiagnosis it is very important to perform a detailed clinicopathologicalcorrelation and a precise immunohistochemicalstudy. Patients and methods: A case in a 76 years oldman with no previous pulmonary pathology is reported. Areview of literature is included

The protein-tyrosine phosphatase CD45 reaches the cell surface via golgi-dependent and -independent pathways.

CD45 is a receptor protein-tyrosine phosphatase essential for T cell development and lymphocyte activation. It is highly glycosylated, with multiple isoforms and glycoforms expressed on the cell surface depending on the cell type and stage of differentiation. Interestingly, we found two pools of newly synthesized CD45 expressed on plasma membrane, one of which arrived by 5 min after synthesis. The remaining pool of CD45 was fully glycosylated and began to arrive at the cell surface at approximately 15 min. The rapidly expressed population of CD45 possessed exclusively endoglycosidase H-sensitive N-linked carbohydrate. Additionally, this rapidly expressed pool of CD45 appeared on the cell surface in a brefeldin A (BFA)-insensitive manner, suggesting that it reached the cell surface independent of the Golgi complex. The remaining CD45 trafficked through the Golgi complex, and transport proceeded via a BFA-sensitive mechanism. These data suggest that CD45 is able to reach the cell surface via two distinct routes. The first is a conventional Golgi-dependent pathway that allows fully processed CD45 to be expressed. The second utilizes an ill defined mechanism that is independent of the Golgi, is BFA-resistant, and allows for the expression of CD45 with immature carbohydrate on the cell surface.

Prevention of adjuvant arthritis by the W3/25 anti-CD4 monoclonal antibody is associated with a decrease of blood CD4(+)CD45RC(high) T cells.

Imbalance between Th1 and Th2 functions is considered to play a key role in the induction and development of several autoimmune diseases, and the correction of that imbalance has led to effective therapies of some experimental pathologies. To examine whether CD4(+)CD45RC(high) (Th1-like) and CD4(+)CD45RC(low) (Th2-like) lymphocytes play a role in the pathogenesis of adjuvant arthritis (AA) and in its prevention by anti-CD4 antibody, CD45RC expression on CD4(+) T cells was determined in arthritic rats and in animals treated with an anti-CD4 MoAb (W3/25) during the latency period of AA. The phenotype of regional lymph node lymphocytes from arthritic rats in the active phase of the disease was determined by flow cytometry. Peripheral blood lymphocytes from rats treated with W3/25 MoAb were also analysed for 2 weeks after immunotherapy finished. IgG2a and IgG1 isotypes of sera antibodies against the AA-inducing mycobacteria, considered to be associated with Th1 and Th2 responses, respectively, were also determined by ELISA techniques. Fourteen days after arthritis induction, regional lymph nodes presented an increase in CD4+CD45RC(high) T cell proportion. Preventive immunotherapy with W3/25 MoAb inhibited the external signs of arthritis and produced a specific decrease in blood CD4(+)CD45RC(high) T cells and a diminution of antibodies against mycobacteria, more marked for IgG2a than for IgG1 isotype. These results indicate a possible role of CD4(+)CD45RC(high) T lymphocytes in the pathogenesis of AA, and suggest that the success of anti-CD4 treatment is due to a specific effect on CD4(+)CD45RC(high) T subset that could be associated with a decrease in Th1 activity.

Markers of cell death-activation in lymphocytes of vertically HIV-infected children naive to highly active antiretroviral therapy: the role of age.

BACKGROUND: Apoptosis plays a major role in depleting CD4(+) lymphocytes during infection with HIV-1. Few data exist on its role during HIV infection of children. Sensitivity of peripheral blood lymphocytes (PBLs) to apoptotic stimuli and the importance of the patient's age remain unclear. OBJECTIVES: We sought to analyze the following: (1) markers of cell death-activation (CD95, CD45 isoforms, and CD28) in PBLs from vertically HIV-infected children of different ages before highly active antiretroviral therapy; (2) changes in other PBL populations; (3) PBL sensitivity to cell death and mitochondrial damages; and (4) role of age during progression of infection. METHODS: Cell culture techniques and flow cytometry were used to analyze surface antigens, PBL susceptibility to apoptosis, or PBL susceptibility to change of mitochondrial membrane potential. RESULTS: Donor age had a strong negative correlation with numbers of CD4(+) and CD8(+) T cells. Virgin T lymphocyte (CD45RA(+), CD95(-)) levels and those of CD95(+) cells showed no correlation with the children's clinical status but did show a correlation with patient age. CD28(-) T lymphocytes were markedly augmented in HIV-infected children but were unrelated to stage of infection or age. A relevant decrease in B lymphocytes and an increase in natural killer cells were also found. Finally, PBLs from HIV-positive children had a marked tendency to undergo apoptosis and mitochondrial damage. CONCLUSION: Changes in PBL phenotype, increased expression of CD95, and high sensitivity to apoptosis suggest that a precocious aging of the immune system occurs in HIV-infected children.

Opioid peptide-expressing leukocytes: identification, recruitment, and simultaneously increasing inhibition of inflammatory pain.

BACKGROUND: Inflammatory pain can be effectively controlled by an interaction of opioid receptors on peripheral sensory nerve terminals with opioid peptides released from immune cells upon stressful stimulation. To define the source of opioid peptide production, we sought to identify and quantify populations of opioid-containing cells during the course of Freund's complete adjuvant-induced hind paw inflammation in the rat. In parallel, we examined the development of stress-induced local analgesia in the paw. METHODS: At 2, 6, and 96 h after Freund's complete adjuvant inoculation, cells were characterized by flow cytometry using a monoclonal pan-opioid antibody (3E7) and antibodies against cell surface antigens and by immunohistochemistry using a polyclonal antibody to beta-endorphin. After magnetic cell sorting, the beta-endorphin content was quantified by radioimmunoassay. Pain responses before and after cold water swim stress were evaluated by paw pressure thresholds. RESULTS: In early inflammation, 66% of opioid peptide-producing (3E7+) leukocytes were HIS48+ granulocytes. In contrast, at later stages (96 h), the majority of 3E7+ immune cells were ED1+ monocytes or macrophages (73%). During the 4 days after Freund's complete adjuvant inoculation, the number of 3E7+ cells increased 5.6-fold (P < 0.001, Kruskal-Wallis test) and the beta-endorphin content in the paw multiplied 3.9-fold (P < 0.05, Kruskal-Wallis test). In parallel, cold water swim stress-induced analgesia increased by 160% (P < 0.01, analysis of variance). CONCLUSIONS: The degree of endogenous pain inhibition is proportional to the number of opioid peptide-producing cells, and distinct leukocyte lineages contribute to this function at different stages of inflammation. These mechanisms may be important for understanding pain in immunosuppressed states such as cancer, diabetes, or AIDS and for the design of novel therapeutic strategies in inflammatory diseases.

Isolation and purification of lymphocyte subsets from gut-associated lymphoid tissue in neonatal swine.

The characterization of lymphoid subsets isolated from different anatomical sites is of great importance for understanding the mechanisms and interactions of normal and pathological immune reactions in the pig. The objective of this study was to standardize a protocol for the isolation of lymphocytes from mucosal tissues of neonatal pigs. Specific protocols for the isolation of lymphocytes from Peyer's patches of jejunum (jejPP) and ileum (ilPP), the Intraepithelial (IE) and lamina propria (LP) compartments of the jejunum and ileum, the mesenteric lymph nodes (MLN), and the peripheral blood (PBMC) are described in detail. The analysis of the cells isolated indicated a high viability (>90%). The histological sections from fragments collected from the intestine demonstrated that in nursing young pigs, the recovery of IE and LP lymphocytes may be limited because of the low numbers of lymphocytes present in early age. In addition, the presence of large intracytoplasmic vacuoles and hyaline droplets between the columnar epithelial cells during the first week of age interferes with the isolation of pure lymphocytes from the IE and LP compartments. Optimal lymphocyte yields for all the samples analyzed was confirmed by immunostaining with the pan-lymphocyte marker, CD45. The successful isolation and comparison of large numbers of pure populations from compartmentalized areas of the intestine and associated lymphoid tissues opens up a broad area for the investigation of mucosal immune responses of pigs.

Protection against virulent Mycobacterium avium infection following DNA vaccination with the 35-kilodalton antigen is accompanied by induction of gamma interferon-secreting CD4(+) T cells.

Mycobacterium avium is an opportunistic pathogen that primarily infects immunocompromised individuals, although the frequency of M. avium infection is also increasing in the immunocompetent population. The antigen repertoire of M. avium varies from that of Mycobacterium tuberculosis, with the immunodominant 35-kDa protein being present in M. avium and Mycobacterium leprae but not in members of the M. tuberculosis complex. Here we show that a DNA vector encoding this M. avium 35-kDa antigen (DNA-35) induces protective immunity against virulent M. avium infection, and this protective effect persists over 14 weeks of infection. In C57BL/6 mice, DNA vaccines expressing the 35-kDa protein as a cytoplasmic or secreted protein, both induced strong T-cell gamma interferon (IFN-gamma) and humoral immune responses. Furthermore, the antibody response was to conformational determinants, confirming that the vector-encoded protein had adopted the native conformation. DNA-35 immunization resulted in an increased activated/memory CD4(+) T-cell response, with an accumulation of CD4(+) CD44(hi) CD45RB(lo) T cells and an increase in antigen-specific IFN-gamma production. The protective effect of the DNA-35 vectors against M. avium infection was comparable to that of vaccination with Mycobacterium bovis BCG and significantly greater than that for previous treated infection with M. avium. These results illustrate the importance of the 35-kDa protein in the protective response to M. avium infection and indicate that DNA vaccination successfully promotes a sustained level of protection during chronic M. avium infection.

Switch in the protein tyrosine phosphatase associated with human CD100 semaphorin at terminal B-cell differentiation stage.

Human CD100, the first semaphorin identified in the immune system, is a transmembrane protein involved in T-cell activation. In the present study, we showed that activation of peripheral blood or tonsillar B lymphocytes induced the expression of CD100 in CD38(+)CD138(-) cell populations, including in CD148(+) subpopulations, thus expressing a memory B-cell-like phenotype. Using an in vitro enzymatic assay, we found that protein tyrosine phosphatase (PTP) activities were immunoprecipitated with CD100 in these cell populations, which were isolated by cell sorting, as well as in most B-cell lines representing various stages of B-cell differentiation. Immunodepletion and Western blotting experiments demonstrated that CD45 was the PTP associated with CD100 in cell lines displaying pre-B, activated B, and pre-plasma cell phenotypes. CD45 also accounted for PTP activity immunoprecipitated with CD100 in CD38(+)CD138(-) cells sorted after activation of peripheral blood or tonsillar B lymphocytes. In contrast, no CD100-CD45 association was observed in plasma cell lines corresponding to the terminal B-cell differentiation stage. CD148, the other transmembrane PTP known to be implicated in lymphocyte signaling pathways, was either only partly involved in the CD100-associated PTP activity or not expressed in plasma cell lines, indicating the association of CD100 with another main PTP. Our data show that CD100 is differentially expressed and can functionally associate with distinct PTPs in B cells depending on their activation and maturation state. They also provide evidence for a switch in the CD100-associated PTP at terminal stage of B-cell differentiation.

Transformation of undifferentiated Thy-1lo B220+ thymic lymphoid cells by the Abelson murine leukemia virus.

Intrathymic injection of the Abelson murine leukemia virus (A-MuLV) results in transformation of immature T and B lymphoid cells. In this report we demonstrate that the concentration of A-MuLV injected into murine thymi influences the selection of the transformation target. Thus, concentrated A-MuLV gives rise to Thy-1+ B220- thymomas. In contrast, dilute virus induces B220+ thymomas that also express low levels of Thy-1 (Thy-1lo), a phenotype that is similar to marrow-derived progenitor B-lymphoid cells (pro-B cells) that are highly susceptible to A-MuLV transformation in vitro. However, rare B220+ lymphoid cells isolated from normal adult thymi were not transformed by A-MuLV in vitro, while B220+ cells isolated from bone marrow were highly susceptible to transformation by A-MuLV. The Thy-1lo B220+ population in the primary thymomas had not rearranged TCRgamma, TCRbeta, or Igkappa genes, but contained subpopulations that assembled Ig DJ(H) or VDJ(H) genes and were therefore similar to transformed pro- and pre-B cells obtained from A-MuLV infected fetal liver and adult bone marrow, respectively. However, unlike A-MuLV-transformed pro- and pre-B cells, many (40-70%) of the Thy-1lo B220+ transformed thymoma cells had not rearranged Igh genes, and therefore appear to represent undifferentiated lymphoid cells. We conclude that A-MuLV may transform an undifferentiated lymphoid target in the thymus.

Mucosal mast cell responses are not required for protection against infection with the murine nematode parasite Trichuris muris.

The involvement of mucosal mast cells (MMC) in protection against infection with the murine nematode parasite Trichuris muris was studied in genetically mast cell-deficient WBB6F1-W/Wv mice and their normal littermates WBB6F1-+/+ mice. Expulsion of T. muris worms occurred in infected +/+ mice, whereas no worm expulsion was observed in infected W/Wv mice where the infection persisted until at least day 46 postinfection. No MMC responses were induced in either infected W/Wv or +/+ mice. Specific IgG1and IgG2a antibodies to T. muris excretory/secretory antigens were observed in infected W/Wv and +/+ mice, and antibody production showed similar kinetics. Interleukin 4 production by concanavalin A (Con A)-stimulated mesenteric lymph node cells (MLNC) was induced preferentially in infected +/+ mice. T. muris infection increased the levels of IFN-gamma produced by Con A-stimulated MLNC of infected W/Wv and +/+ mice, with the levels of IFN-gamma in infected W/Wv mice being higher than those in infected +/+ mice. Taken together, these results indicate that W/Wv and +/+ mice are susceptible and resistant to T. muris infection, respectively, and that MMC responses are not required for protective immunity.

Cell-specific glycoforms of sialoadhesin and CD45 are counter-receptors for the cysteine-rich domain of the mannose receptor.

We previously reported that CR-Fc, an Fc chimeric protein containing the cysteine-rich (CR) domain of the mannose receptor, binds to marginal zone metallophilic macrophages (Mo) and B cell areas in the spleen and to subcapsular sinus Mo in lymph nodes of naive mice (CR-Fc(+) cells). Several CR-Fc ligands were found in spleen and lymph node tissue lysates using ligand blots. In this paper we report the identification of two of these ligands as sialoadhesin (Sn), an Mo-specific membrane molecule, and the leukocyte common antigen, CD45. CR-Fc bound selectively to Sn purified from spleen and lymph nodes and to two low molecular weight isoforms of CD45 in a sugar-dependent manner. CR-Fc binding and non-binding forms of Sn, probably derived from CR-Fc(+) and CR-Fc(-) cells respectively, were selected from spleen lysates. Analysis of the glycan pool associated with the CR-Fc-binding form revealed the presence of charged structures resistant to sialidase, absent in the non-binding form, that could correspond to sulfated structures. These results confirm the identification of the CR region of the mannose receptor as a lectin. We also demonstrate that the same glycoprotein expressed in different cells of the same organ can display distinct sugar epitopes that determine its binding properties.

Detection of viral antigen in placenta and fetus of cattle acutely infected with bovine viral diarrhea virus.

The reproductive organs and fetuses of seven Norwegian Red heifers were investigated for the presence of bovine viral diarrhea virus (BVDV) antigen during the time of initial transplacental transmission of the virus. The heifers were inoculated with a noncytopathogenic BVDV at day 85/86 of gestation and were slaughtered at day 7, 10, 14, 18, or 22 postinoculation (pi). Cryostat sections of uterus, ovaries, placentomes, intercotyledonary fetal membranes, and fetal organs were examined using immunohistochemical techniques. A double immunofluorescence technique was used to identify cells that showed staining with antibodies against the leukocyte common antigen CD45 or the intermediate filament vimentin and BVDV antigens. The earliest stage of infection at which BVDV antigen could be detected in the fetuses was 14 days pi. At this stage, BVDV antigen was detected in cells of mesenchymal origin in the lungs and in large cells that morphologically resembled immature megakaryocytes in the liver. In the intercotyledonary fetal membranes and in the placentomes, BVDV antigen was not detected until 18 and 22 days pi, respectively. BVDV antigen was not detected in maternal tissue from any of the heifers. The present results indicate that fetal infection with BVDV can take place without preceding or simultaneous high concentrations of BVDV in uterus or placenta of acutely infected heifers.

A model for the origin of TCR-alphabeta+ CD4-CD8- B220+ cells based on high affinity TCR signals.

The origin of TCR-alphabeta+ CD4-CD8- cells is unclear, yet accumulating evidence suggests that they do not represent merely a default pathway of unselected thymocytes. Rather, they arise by active selection as evidenced by their absence in mice lacking expression of class I MHC. TCR-alphabeta+ CD4-CD8- cells also preferentially accumulate in mice lacking expression of Fas/APO-1/CD95 (lpr) or Fas-ligand (gld), suggesting that this subset might represent a subpopulation destined for apoptosis in normal mice. Findings from mice bearing a self-reactive TCR transgene support this view. In the current study we observe that in normal mice, TCR-alphabeta+ CD4-CD8- thymocytes contain a high proportion of cells undergoing apoptosis. The apoptotic subpopulation is further identified by its expression of B220 and IL2Rbeta and the absence of surface CD2. The CD4-CD8- B220+ phenotype is also enriched in T cells that recognize endogenous retroviral superantigens, and can be induced in TCR transgenic mice using peptide/MHC complexes that bear high affinity, but not low affinity, for TCR. A model is presented whereby the TCR-alphabeta+ CD2- CD4-CD8- B220+ phenotype arises from high intensity TCR signals. This model is broadly applicable to developing thymocytes as well as mature peripheral T cells and may represent the phenotype of self-reactive T cells that are increased in certain autoimmune conditions.

Autoantibodies to CD45 in systemic lupus erythematosus.

Western blotting with U937 cell extracts as the substrate, and enzyme-linked immunosorbent assays (ELISA) with U937-, Jurkat- and Daudi cell-purified CD45 molecules were used to detect anti-CD45 reactivity in patients with systemic lupus erythematosus (SLE). By immunoblotting, 16 of 64 SLE sera were shown to be positive (25.0%). In the ELISAs, 13 out of 18 SLE sera reacted with the target CD45. Of these, three were not detectable on the blot. Importantly, 12 of these ELISA-positive sera contained IgM and IgG auto-antibodies. Neuraminidase-treatment of U937-precipitated CD45 molecules enhanced the reactivity to most of the isoforms, indicating that the antibodies may bind to asialylated polysaccharides.

Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny.

We have investigated the developmental relationship of the hemopoietic and endothelial lineages in the floor of the chicken aorta, a site of hemopoietic progenitor emergence in the embryo proper. We show that, prior to the onset of hemopoiesis, the aortic endothelium uniformly expresses the endothelium-specific membrane receptor VEGF-R2. The onset of hemopoiesis can be determined by detecting the common leukocyte antigen CD45. VEGF-R2 and CD45 are expressed in complementary fashion, namely the hemopoietic cluster-bearing floor of the aorta is CD45(+)/VEGF-R2(-), while the rest of the aortic endothelium is CD45(-)/VEGF-R2(+). To determine if the hemopoietic clusters are derived from endothelial cells, we tagged the E2 endothelial tree from the inside with low-density lipoproteins (LDL) coupled to DiI. 24 hours later, hemopoietic clusters were labelled by LDL. Since no CD45(+) cells were inserted among endothelial cells at the time of vascular labelling, hemopoietic clusters must be concluded to derive from precursors with an endothelial phenotype.

CD45 and RPTPalpha display different protein tyrosine phosphatase activities in T lymphocytes.

To examine the substrate specificity and function of two receptor protein tyrosine phosphatases, CD45 and RPTPalpha, RPTPalpha was expressed in a CD45(-), T-cell receptor (TCR)+, BW5147 T-lymphoma cell. High levels of expression of RPTPalpha did not fully restore either proximal or distal TCR-mediated signalling events. RPTPalpha was unable to reconstitute the phosphorylation of CD3zeta and did not increase the expression of the activation marker, CD69, on stimulation with TCR/CD3. RPTPalpha did not significantly alter the phosphorylation state or kinase activity of two CD45 substrates, p56(lck) or p59(fyn), suggesting that RPTPalpha does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTPalpha was approx. one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed in vitro with purified recombinant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56(lck) as substrates indicated that CD45 was consistently more active than RPTPalpha, having both higher Vmax and lower Km values. Thus CD45 is intrinsically a much more active phosphatase than RPTPalpha, which provides one reason why RPTPalpha cannot effectively dephosphorylate p56(lck) and substitute for CD45 in T-cells. This work establishes that these two related protein tyrosine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase activity.