Phenotypic and functional characterization of cytotoxic cells derived from endomyocardial biopsies in human cardiac allografts.

This study was undertaken to characterize the phenotype and function of lymphocytes derived from endomyocardial biopsies in heart transplant patients. To this aim, tissue infiltrating lymphocytes were derived from seven heart transplant patients and were analyzed for the expression of a panel of markers, including CD3, CD4, CD8, CD16, CD56, CD45RA, CD45RO, alpha/beta and gamma/delta T cell receptor, and for their ability to lyse a series of targets, including NK-sensitive K-562 targets, NK-resistant Raji targets, donor related, and unrelated normal splenocytes. Our data show that the majority of cultured lymphocytes expressed the CD3+ phenotype and the alpha/beta T cell receptor. The CD4 and CD8 molecules were heterogeneously expressed among T cell lines tested. Concerning cytotoxic related markers, a significant percentage of cells were CD56+. The evaluation of CD45 isoforms showed that both "naive" and "memory" cells were present among heart TIL. Cytotoxic in vitro studies demonstrated that all our T cell lines showed an efficient cytotoxic machinery when tested against NK-sensitive targets. A marked lysis of donor-related splenocytes was demonstrated in all patients tested. To investigate the role of CD3 and HLA class I molecules in the cytotoxic mechanisms taking place in human heart allograft rejection mechanisms, TIL were assessed for their lytic activity against different targets in the presence of anti-CD3 and anti-HLA class I monoclonal antibodies (mAbs). Although donor-specific cytotoxicity was considerably inhibited by the anti-CD3 mAb, no inhibitory effect was displayed by this antibody on TIL-mediated cytotoxicity against donor-unrelated splenocytes. Anti-HLA class I mAb was able to inhibit both allospecific and nonallospecific cytotoxicity. These data suggest that different types of cytotoxic cells may be propagated from biopsy specimens of heart transplant patients.     

Phenotypic and functional characterization of NK cells in human immune response against the dimorphic fungus Paracoccidioides brasiliensis

Besides their role in fighting viral infection and tumor resistance, recent studies have shown that NK cells also participate in the immune response against other infectious diseases. The aim of this study was to characterize the possible role of NK cells in the immune response against Paracoccidioides brasiliensis. Purified NK cells from paracoccidioidomycosis patients and healthy individuals were incubated with P. brasiliensis yeast cells or P. brasiliensis-infected monocytes, with or without the addition of recombinant IL-15. We found that NK cells from paracoccidioidomycosis patients exhibit a lower cytotoxic response compared with healthy individuals. NK cells are able directly to recognize and kill P. brasiliensis yeast cells, and this activity seems to be granule-dependent but perforin-independent, whereas the cytotoxicity against P. brasiliensis-infected monocytes is perforin-dependent. These results indicate that NK cells participate actively in the immune response against the P. brasiliensis infection either by directly destroying yeast cells or by recognizing and killing infected cells. Granulysin is the possible mediator of the cytotoxic effect, as the reduced cytotoxic activity against the yeast cells detected in patients with paracoccidioidomycosis is accompanied by a significantly lower frequency of CD56(+)granulysin(+) cells compared with that in healthy controls. Furthermore, we show that NK cells released granulysin in cultures after being stimulated by P. brasiliensis, and this molecule is able to kill the yeast cells in a dose-dependent manner. Another important finding is that stimulated NK cells are able to produce proinflammatory cytokines (IFN-γ and TNF-α) supporting their immunomodulatory role in the infection.     

Phenotypic characteristics of heat-resistant murine neuroblastoma cells

One-step selection of murine neuroblastoma (N18TG2 cell line) for the resistance to injuring action of higher temperature resulted in obtaining cell line NTR1 stably capable of proliferating at 40 degrees C. The thermoresistance is shown to be coupled with the changes in some phenotypic features of NTR1 cells: the multiplication rate, saturation density of cells, morphological features, inducibility of long neurite-like processes and adhesion. A possible significance of plasma membrane changes in genetically stable thermoresistance of NTR1 neuroblastoma cells is discussed.     

Phenotypic characteristics of thermotolerantCampylobacter from human and animal sources

Five reference strains and 314 field strains ofCampylobacter growing at 42°C, but not at 25°C, were characterized by tests of hippurate hydrolysis and sensitivity to nalidixic acid, to metronidazole, and to 2,3,5-triphenyltetrazolium chloride (TTC). All strains but seven were TTC-resistant by the disc test used. Of 168 human isolates, 87% hydrolyzed hippurate; three of these strains were nalidixic acid-resistant. Also hippurate-positive were all 23 strains from ovine abortion, 79% of 43 avian strains, two of six bovine isolates, and one of two strains of equine origin. Seventy-two porcine strains were all hippurate-negative. Metronidazole sensitivity was found to be a variable property in hippurate-positive strains, although present in all but four hippurate-negative strains. A group of eight nalidixic acid-resistant, hippurate-negative isolates of porcine and bovine origin probably represent a new group ofCampylobacter.     

Phenotypic and functional characteristics of in vivo-activated Langerhans cells

After short term culture (2 to 3 days), Langerhans cells (LC) exhibit increased class II MHC Ag and become more potent APC than freshly obtained LC in primary allogeneic and syngeneic T cell activation. To determine whether in vivo LC undergo changes similar to cultured LC, we examined the phenotypic and functional characteristics of LC harvested from ear skin of naive mice painted with various haptens and primary irritants. At 24 h after application of 3% trinitrochlorobenzene, LC appear larger and exhibit more intense staining in epidermal sheets using anti-I-A antibodies, and there was a two- to threefold increase in I-A and I-E expression by LC using flow microfluorimetry analysis. CD45 Ag expression was not altered. Flow microfluorimetry profiles showed the presence of two different LC populations based on fluorescence intensity, i.e., one with the same Ia density as nontreated LC and the other (representing 22 to 50% of all LC) with a markedly enhanced Ia density, (i.e., a 10-fold increase in I-A and I-E). This phenotypic change was observed only with haptens, such as trinitrochlorobenzene, dinitrofluorobenzene, oxazolone, and cinnamic aldehyde. In contrast, application of 10 to 30% sodium lauryl sulfate or vehicle controls did not induce this change. Functionally, LC obtained from hapten-painted mice induced a two- to fivefold increase in 3[H]-TdR incorporation by syngeneic or allogeneic T cells, compared to equal numbers of LC from nontreated or vehicle-treated or sodium lauryl sulfate-treated mice. These phenotypic and functional changes that occur in vivo are therefore analogous to those seen when LC are cultured for short periods of time. Thus, activated LC appear in vivo in response to the epicutaneous application of haptens and may represent an essential step in hapten-specific sensitization.     

Phenotypic and functional characteristics of porcine peritoneal mesothelial cells

The objective of this study was to establish a technique to isolate porcine mesothelial cells (PMCs) from omental tissue and to compare them to human mesothelial cells (HMCs). The PMCs were dispersed by collagenase digestion and isolated on a Ficoll layer. Their morphologic and ultrastructural features were assessed at confluence by light and electronic microscopy, and they were characterized by immunohistochemistry using specific HMC markers. PMC proliferation was studied in the presence of growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or transforming growth factors beta1, beta2, or beta3 (TGF). Fibrinolytic PMC activity was detected by zymography for tissue plasminogen activator (tPA) and by reverse zymography for plasminogen activator inhibitor-1 (PAI-1). The recalcification time of cell lysates was used to define PMC procoagulant activity, and gelatinase zymography was used to detect metalloproteinase production. At confluence, PMCs formed typical cobblestone monolayers and exhibited structural features characteristic of HMCs. Weibel Palade bodies were never seen. Specific HMC markers (HBME1, ME1, WT1) cross-reacted with PMCs. As HMCs and PMCs coexpressed cytokeratin and vimentin, and also expressed vinculin and alpha-actin. Addition of PDGF or EGF to the culture medium stimulated PMC proliferation. PMCs constitutively expressed fibrinolytic and procoagulant activity and secreted MMP9 and MMP2. The technique described in this study allows isolation of mesothelial cells from porcine omental tissue. These porcine cells exhibit a mesothelial phenotype and functional properties similar to those of HMCs. Our data warrant an evaluation of mesothelial cells as targets in several therapeutic strategies with porcine models.     

Phenotypic characteristics of human monocytes undergoing transendothelial migration

In our study we characterised the immunophenotype of monocytes that migrated through an endothelial cell (EC) monolayer in vitro. We found that monocyte migration led to an enhanced expression of CD11a, CD33, CD45RO, CD54 [intercellular cell-adhesion molecule (ICAM)-1] and human leucocyte antigen-DR. The most striking increase was observed for ICAM-1 when ECs were activated with tumour necrosis factor-α and interleukin-1α. The results of our study indicate the following: (1) there is a characteristic immunophenotype on the surface of monocytes after transendothelial migration; (2) this phenotype seems to be induced by interactions between monocytes and ECs; and (3) this change is enhanced by the pretreatment of ECs with cytokines. Taken together, the results suggest that local cytokine production activating ECs is sufficient to enhance monocyte migration and that this, in turn, can induce changes consistent with an activated phenotype known to be interactive between antigen-presenting cells and T cells. These results have implications for our pathogenetic insights into rheumatoid arthritis.     

Phenotypic and functional characteristics of human newborns' B lymphocytes.

It has been demonstrated two major facts concerning human newborns' B lymphocytes: 1) they differentiate poorly into Ig-producing cells and 2) they express CD5 and CD1c membrane proteins. We have further analyzed human newborns' B cell characteristics and found that approximately half of them express activation Ag, i.e., 4F2 and IL-2R, both associated in significant proportions with CD23 and Bac-1. These membrane Ag were found both on CD5(+) and CD5(-) B cells. Newborns' B cells do not exhibit other activation markers because they express surface IgD, and because their size, RNA, and DNA contents do not differ from those of adults' B cells, indicating that they are in the G0/G1 cell cycle phase. Newborns' B cell proliferation can be induced by rIL-2, rIL-4, low m.w. B cell growth factor, and by Staphylococcus aureus protein A. It is presently difficult to build a hypothesis accounting for all the specific findings made on newborns' B cells. It is not known for instance whether CD5(+) and (-) B cells belong to distinct subsets as suggested by the fluorescence intensity curve obtained with an anti-CD5 antibody or to distinct stages in a unique pattern of B cell maturation during fetal and newborn life. This may indicate that partially activated B cells actually produce natural polyspecific autoantibodies of the IgM isotype found in newborns' human serum.     

Phenotypic and functional characteristics of spermatogonial stem cells in rats

Spermatogonial stem cells (SSCs) are at the foundation of the highly productive spermatogenic process that continuously produces male gametes throughout postnatal life. However, experimental evaluation of SSCs in postnatal testes is complicated because these cells are extremely rare and few defining morphology or biochemical characteristics are known. In this study, we used the spermatogonial transplantation functional assay, combined with fluorescence-activated cell sorting (FACS) analysis to identify cellular, biochemical and surface antigenic characteristics of SSCs in rat testes during development. Our results demonstrated that forward scatter (FSc)(hi), side scatter (SSc)(hi), mitochondria membrane potential (DeltaPsim)(lo), Ep-CAM(+), Thy-1(+), beta3-integrin(+) stem cells in neonate rat testes become SSc(lo), DeltaPsim(hi), Ep-CAM(+), Thy-1(lo), beta3-integrin(-) stem cells in pup rat testes. Furthermore, prospective identification of rat testis cell populations (Ep-CAM(+)), highly enriched for SSCs (1 in 13 for neonate; 1 in 8.5 for pup) enabled us to predict the Thy-1 and beta3-integrin status of stem cells in neonate and pup testes, which was subsequently confirmed by transplantation analyses. Systematic characterization of SSCs enabled the production of testis cell populations highly enriched (up to 120-fold) for SSCs and will facilitate future investigations of functional and genomic characteristics.     

Phenotypic characteristics of peripheral blood T cells regulating colony growth by nontransformed human B lymphocytes

The phenotypic characteristics of peripheral blood T cell subpopulations regulating human B cell colony growth stimulated by Staph protein A were investigated. Colony growth was facilitated by OKT4 cells, and T cells expressing DR antigens were found to be partially responsible for colony facilitation. A linear increase in the magnitude of colony growth was observed with greater T cell numbers, and maximal colony enhancement occurred when T cells were present during the early stages of colony formation. OKT8 cells did not enhance colony growth and also inhibited the facilitation of colony formation by OKT4 cells. Other experiments showed that the functional activities of OKT4 and OKT8 cells differed in their requirements for DNA synthesis. Although active T cell DNA synthesis was absolutely required for the facilitation of colony growth at all concentrations tested, DNA synthesis was not needed for OKT8 inhibition of OKT4 promotion of colony formation. Thus, distinct T cell subsets whose functional properties differ in their requirements for DNA synthesis regulate human colony growth.     

Phenotypic characteristics of natural killer cells in acute hepatitis

Natural killer (NK) cells are the principal effector cell population in innate immune defense against many types of infections. These cells are enriched in the liver, where they comprise approximately 40% to 60% of the intrahepatic lymphocyte pool compared to the peripheral blood compartment. In chronic HBV and HCV infection, NK cells were reported to be partially dysfunctional due to impaired IFN-Γ secretion. Few studies have examined phenotypic features of NK cells in acute hepatitis. We identified NK (CD56+CD3-) cell populations in patients with acute hepatitis by examining the expressions of phenotypic NK cell markers (CD16, NKG2A, and NKG2D). Peripheral blood mononuclear cells were isolated from patients with acute hepatitis A (7) and patients with non-viral acute toxic hepatitis (6) during the symptomatic and convalescent phases. Expressions of NK (CD56+CD3-) cell markers, CD16, NKG2A, and NKG2D, were measured by flow cytometry. Symptomatic acute hepatitis including non-viral hepatitis and HAV infection showed significant increases of NKG2A expression compared to healthy controls. Interestingly, there was a direct correlation between the proportion of NK cell populations and liver function parameters (AST, ALT) in HAV infection. The strong correlation was also observed between the expression of NKG2A+NK cells and ALT, which suggests that most of NK cells in severe phase of disease express high level of NKG2A on their surface. In addition, decreased number of NK cells (CD56+CD3-) in symptomatic phase began to increase in the convalescent phase of acute hepatitis A. However, the expression of NKG2A tended to be reduced, which indicates that NKG2A, the inhibitory receptor on NK cells, can be a severity parameter in acute hepatitis.     

Phenotypic switching of Cryptococcus neoformans can influence the outcome of the human immune response

The human pathogenic fungus Cryptococcus neoformans exhibits the phenomenon of phenotypic switching, a process that generates variant colonies that can differ in morphology, virulence and other characteristics such as capsular glucuronoxylomannan (GXM) size and structure. A previous study established that mucoid colony (MC) variants of C. neoformans were more virulent and elicited a different inflammatory response than smooth colony (SM) variants. In this study, we investigated the interaction of cells from MC and SM variants and their respective GXMs with human T cells and monocytes. Specifically, we measured CD40, CD80 and CD86 expression, lymphoproliferation and interleukin (IL)-4, IL-10, interferon (IFN)-gamma and IL-12Rbeta2 expression in the presence and absence of variant cells and their GXMs. For some immune parameters, both MC and SM strains produced similar results, in particular no differences were observed in IL-4 induction. However, for other critical parameters, including CD86 expression, lymphoproliferation and IL-10 production, the MC variant had effects that can be expected to impair the immune response. Hence, a single C. neoformans strain can elicit several different immune responses depending on the colony type expressed, and this is unlikely to be accounted for by differences in phagocytosis only. The results provide a potential explanation for the higher virulence of the MC variant based on the concept that these cells inhibit the development of a vigorous immune response. Furthermore, the results suggest a mechanism by which phenotypic switching can generate variants able to evade the immune response.     

Phenotypic expression of human hepatoma cells in culture

Hepatomas thrive in a hypoxic environment resulting in the induction of a cluster of hypoxia related genes. The protein phenotypic expression include hypoxia inducible factor-alpha, prolyl-4-hydroxylase, vascular endothelear growth factor and erythropoietin. The present study was undertaken to determine if human hepatoma cells when cultured for 72 h in the presence of serum under normoxia would maintain their cancerous phenotypic expression of certain hypoxia inducible genes. Our positive results affords an in vitro model system to test hypoxia inhibitors on the expression and the intracellular compartmentalization or the secretion of these hypoxia-inducible proteins.