Differential effects of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells.

Bipotential T/natural killer (NK) progenitor cells are destined to differentiate mainly into T cell receptor (TCR) alpha beta and TCR gamma delta cells in a thymic microenvironment, whereas extrathymically they selectively develop into NK cells. The exact environmental conditions that are required for differentiation into these three leukocyte populations are largely unknown. In this report, we have investigated and compared the effect of interleukin (IL)-15 and IL-2 in this process. The IL-15 receptor is composed of the gamma and beta chains of the IL-2 receptor (IL-2R gamma and IL-2R beta) and of a specific alpha chain (IL-15R alpha). Here, it is shown that IL-15 mRNA is mainly expressed in thymic epithelial stromal cells, whereas IL-2 mRNA is exclusively expressed in thymocytes. IL-2R beta-expressing cells were present in the fetal thymus with a CD25-CD44+Fc gamma R+HSA-/low TCR- phenotype, which is characteristic of progenitor cells. These cells also expressed IL-15R alpha messenger RNA. Sorted IL-2R beta + TCR- cells differentiated into TCR alpha beta and TCR gamma delta cells after transfer to alymphoid thymic lobes, whereas culture of the same sorted cells in cell suspension in the presence of IL-15 resulted in the generation of functional NK cells. This shows that IL-2R beta +TCR- cells of the fetal thymus contain bipotential T/NK progenitors. Addition of low concentrations of IL-15 to fetal thymic organ culture (FTOC) resulted in an increase of all T cell subpopulations. The largest expansion occurred in the TCR gamma delta compartment. In contrast, low concentrations of IL-2 did not result in a higher total cell number and did not induce outgrowth of TCR gamma delta cells. High concentrations of IL-15 blocked TCR alpha beta development and shifted differentiation towards NK cells. Differentiation towards TCR gamma delta cells still proceeded. High concentrations of IL-2 similarly induced development into NK cells, but the cell number was fourfold lower than in IL-15 cultures. Importantly, blocking of IL-2R alpha in IL-2-treated FTOC resulted in a drastic increase in cell number, indicating that IL-2R alpha negatively regulates cell expansion. Collectively, these experiments provide direct evidence that IL-15 and IL-2 differentially affect the differentiation of bipotential T/NK progenitors.

Intrathymic differentiation of V gamma 3 T cells.

Whereas there is considerable information on the phenotypic and functional maturation of T cell receptor (TCR) alpha/beta thymocytes, comparatively little is known of the maturational processes that affect development of TCR-gamma/delta thymocytes. One class of gamma/delta T cells, those bearing the V gamma 3 gene product, are generated only during the early fetal stages of thymic development, and then migrate to the skin. Here we examine the intrathymic differentiation of these V gamma 3+ cells. The earliest V gamma 3 cells to appear in the thymus expressed low levels of TCR (V gamma 3low) and high levels of heat stable antigen (HSA). Over the next few days, V gamma 3+ thymocytes appeared which expressed high levels of TCR (V gamma 3high) and very low levels of HSA. The antigens CD5, CD45RB, and MEL14 were also differentially expressed on V gamma 3low versus V gamma 3high thymocytes, but the shift in expression was the opposite as compared with immature and mature TCR-alpha/beta thymocytes. Transfer experiments of sorted V gamma 3low/HSAhigh thymocytes to SCID thymic lobes showed that these cells were indeed the precursors of V gamma 3high/HSAlow thymocytes. The phenotype of the V gamma 3high thymocytes was similar to that of the postthymic V gamma 3+ cells found in the skin of adult mice. The differentiation of V gamma 3low in V gamma 3high thymocytes was also observed in fetal thymic organ culture. Addition of cyclosporin A (CsA) to these cultures had little effect on the appearance of V gamma 3low/HSAhigh cells, but blocked the appearance of V gamma 3high/HSAlow cells. These results show that, like alpha/beta T cells, V gamma 3+ thymocytes differentiate from TCRlow precursors to cells with a mature phenotype and that CsA inhibits this transition.

T-, B- and NK-lymphoid, but not myeloid cells arise from human CD34(+)CD38(-)CD7(+) common lymphoid progenitors expressing lymphoid-specific genes.

Hematopoietic stem cells in the bone marrow (BM) give rise to all blood cells. According to the classic model of hematopoiesis, the differentiation paths leading to the myeloid and lymphoid lineages segregate early. A candidate 'common lymphoid progenitor' (CLP) has been isolated from CD34(+)CD38(-) human cord blood cells based on CD7 expression. Here, we confirm the B- and NK-differentiation potential of CD34(+)CD38(-)CD7(+) cells and show in addition that this population has strong capacity to differentiate into T cells. As CD34(+)CD38(-)CD7(+) cells are virtually devoid of myeloid differentiation potential, these cells represent true CLPs. To unravel the molecular mechanisms underlying lymphoid commitment, we performed genome-wide gene expression profiling on sorted CD34(+)CD38(-)CD7(+) and CD34(+)CD38(-)CD7(-) cells. Interestingly, lymphoid-affiliated genes were mainly upregulated in the CD7(+) population, while myeloid-specific genes were downregulated. This supports the hypothesis that lineage commitment is accompanied by the shutdown of inappropriate gene expression and the upregulation of lineage-specific genes. In addition, we identified several highly expressed genes that have not been described in hematopoiesis before.

WITHDRAWN: Immunohistochemical localization of Ih channel HCN3 in the rat brain.

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Expression and function of Fc receptors in the thymus.

In this review, the expression and function of Fc receptors (FcRs) in the thymus is discussed. In the murine thymus, Fc gamma RII and Fc gamma RIII are expressed on early thymocyte precursors, which can differentiate in both T and NK cells. TCR alpha beta thymocytes that are differentiating along the CD4-CD8 pathway do not express Fc gamma Rs any longer. Mature CD4-CD8 double negative TCR alpha beta and TCR V gamma 3 cells, however, constitutively express Fc gamma RII/III. The generation of gene-deficient mice has shown that neither Fc gamma Rs nor Fc epsilon RII are indispensable for murine thymus-dependent T cell development, whereas normal development of thymus-independent peripheral T cells is dependent on the presence of the FcR gamma chain. In the human thymus, a low number of CD3-CD4-CD8 triple negative cells expresses Fc gamma RIII, but these cells are mainly NK cells. Fc epsilon RII is expressed on human thymic epithelial cells. Although the unaltered thymic development of T cells in FcR-deficient mice argues against a fundamental role of FcRs in this process, recent demonstration of FcR ligands of non-immunoglobulin nature in the thymus indicates that the interaction between FcRs and their ligands in the thymus might influence T cell development.

Thymic and extrathymic T cell development.

The majority of T cells located in peripheral blood, spleen and lymph nodes are dependent on the thymus for proper differentiation and function. Only a minority of T lymphocytes located in these lymphoid organs is thymic-independent. In contrast, a large number of thymus-independent T cells is present in the gut epithelium. This review deals with phenotypic and functional characteristics of T cell development, summarizes the knowledge on the cytokine requirement in this process and describes positive and negative selection. The differences between thymic-dependent and thymic-independent T cells are emphasized, including selection processes, CD4-CD8 expression and the composition of the CD3 complex.

Homeobox gene expression profile in human hematopoietic multipotent stem cells and T-cell progenitors: implications for human T-cell development.

Class I homeobox (HOX) genes comprise a large family of transcription factors that have been implicated in normal and malignant hematopoiesis. However, data on their expression or function during T-cell development is limited. Using degenerated RT-PCR and Affymetrix microarray analysis, we analyzed the expression pattern of this gene family in human multipotent stem cells from fetal liver (FL) and adult bone marrow (ABM), and in T-cell progenitors from child thymus. We show that FL and ABM stem cells are similar in terms of HOX gene expression, but significant differences were observed between these two cell types and child thymocytes. As the most immature thymocytes are derived from immigrated FL and ABM stem cells, this indicates a drastic change in HOX gene expression upon entry into the thymus. Further analysis of HOX-A7, HOX-A9, HOX-A10, and HOX-A11 expression with specific RT-PCR in all thymocyte differentiation stages showed a sequential loss of 3' region HOX-A cluster genes during intrathymic T-cell development and an unexpected expression of HOX-A11, previously not recognized to play a role in hematopoiesis. Also HOX-B3 and HOX-C4 were expressed throughout thymocyte development. Overall, these data provide novel evidence for an important role of certain HOX genes in human T-cell development.

Developmentally regulated responsiveness to transforming growth factor-beta is correlated with functional differences between human adult and fetal primitive hematopoietic progenitor cells.

Important functional differences exist between primitive CD34++ CD38- hematopoietic progenitor cells derived from human fetal liver (FL) and adult bone marrow (ABM). FL progenitors are known to have higher proliferative capacities and lower cytokine requirements than their ABM counterparts. In this study, we isolated FL and ABM CD34++ CD38- cells and used a two-stage culture system to investigate the effects of transforming growth factor-beta (TGF-beta) and blocking anti-TGF-beta antibodies (anti-TGF-beta) on these cells. First, we demonstrate that FL progenitors are significantly less sensitive to the inhibitory effects of TGF-beta than ABM cells. Second, whereas ABM cells are significantly stimulated by anti-TGF-beta, only very limited effects are seen on FL cells. Third, we show that the effect of anti-TGF-beta is mainly situated at the level of the initial cell cycles of very primitive progenitor cells with a high proliferation potential. Fourth, we demonstrate that blocking the effects of endogenous TGF-beta reduces the growth factor requirements of ABM cells in order to proliferate and differentiate. Based on these data, we hypothesize that at least part of the functional differences that exist between adult and fetal stem cells can be accounted for by a developmental different responsiveness to TGF-beta.

Biocompatibility parameters in in-vitro simulated automated versus continuous ambulatory peritoneal dialysis.

BACKGROUND: In peritoneal dialysis, the usage of automated peritoneal dialysis (APD) has been steadily increased. As APD means larger volumes of solution and more frequent contact times with fresh dialysate, an additive negative impact on biocompatibility data, exceeding the known effect of conventional PD fluids, seems possible. For an in-vitro comparison of APD and CAPD, a new cell culture system has recently been established. METHODS: A double chamber cell culture system with human mesothelial cells on top of a permeable membrane and growth medium beyond was used for mimicking CAPD and APD. Reflecting the in vivo equilibration pattern, we compared an eight-hour CAPD with a CCPD setting, using a conventional PD solution. Cell viability was assessed with a MTT assay and cell function via constitutive and stimulated IL-6 release. CA125 was measured as a parameter of mesothelial cell integrity, and TGF-1beta was measured as an index of induction of fibrosis. RESULTS: Both the CAPD and the CCPD mode resulted in a significantly lower MTT assay and stimulated IL-6 release compared to growth medium. TGF-1beta and CA125 release did not differ between the PD modes and control. The CAPD and the CCPD mode itself did not differ with regard to MTT assay, IL-6 release, TGF-1beta and CA125 generation. CONCLUSION: From the in-vitro model imitating the acute exposure of mesothelial cells with conventional PD fluid in a CCPD and CAPD mode, there is no evidence that APD, due to the larger volumes of solution and more frequent contact times with fresh dialysate, has an acute, additive negative impact on biocompatibility parameters indicative for peritoneal host defense, mesothelial cell integrity and peritoneal fibrosis.

Murine fetal natural killer cells are functionally and structurally distinct from adult natural killer cells.

Natural killer (NK) cell phenotype and activity was studied by analyzing uncultured and short-time-cultured murine NK cells from fetal day 17 spleen and thymus. In contrast to NK cells from adult mice, freshly sorted fetal NK cells did not contain NK receptor transcripts for Ly-49A, B, C/I, D, F, G2, or H. The only NK receptor transcripts that could be detected were Ly-49E and CD94. It is important that Ly-49E was present at a 10- to 30-fold higher level compared with uncultured NK cells from adult mice. After short-time interleukin-2 culture, the level of Ly-49E mRNA was comparable between fetal and adult NK cells. Functionally, fetal NK cells only killed MHC class I-negative tumor cells when activating NK receptors were cross-linked with antibody. We show that fetal NK cells are mature but are different from NK cells in adult mice regarding their NK cell receptor repertoire and function.

CD34++ CD38- and CD34+ CD38+ human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source.

CD34++ CD38- and CD34+ CD38+ hematopoietic progenitor cells (HPCs) from human fetal liver (FL), cord blood (CB), and adult bone marrow (ABM) were isolated and investigated for their growth characteristics, cytokine requirements and response to two modulators of early hematopoiesis, interferon (IFN)-gamma and macrophage inflammatory protein (MIP)-1alpha. We observed first that a significantly lower percentage of CD34++ cells were CD38- in ABM than in FL and CB. Second, the functional differences between CD34++ CD38- and CD34+ CD38+ cells were less pronounced in FL and CB than in their ABM counterparts. Third, an inverse correlation was found between growth factor response and the ontogenic age of HPCs, and a direct correlation was noted between cytokine requirements and the ontogenic age of HPCs. Fourth, spontaneous colony formation in a classic semisolid culture system was reproducibly obtained only in the ontogenically earliest cells, that is, in FL but not in CB and ABM, in which no such spontaneous colony formation was observed. Fifth, the modulatory effects of IFN-gamma and MIP-1alpha were qualitatively different depending on the ontogenic age of the progenitor source: whereas IFN-gamma was only a selective inhibitor of primitive CD34++ CD38- ABM progenitor cells, it inhibited both CD34++ CD38- and CD34+ CD38+ FL and CB cells to the same extent. In contrast to the effects of MIP-1alpha on ABM, we could not find any consistently stimulatory or inhibitory effects on FL and CB progenitors. In conclusion, important functional and biologic differences exist between FL, CB, and ABM progenitor cells, and these differences could have major implications for the use of these cell populations in preparative protocols of ex vivo expansion, transplantation strategies, or gene transfer experiments.

Evaluation of proviral copy number and plasma RNA level as early indicators of progression in HIV-1 infection: correlation with virological and immunological markers of disease.

OBJECTIVE: We compared the proviral DNA level in peripheral blood mononuclear cells (PBMC), viral RNA level in plasma, presence of p24 antigen in serum, viral phenotype, and results of immunological markers of HIV-1 disease. METHODS: Consecutive samples of 62 HIV-1-infected patients, representing all stages of disease were tested for proviral DNA in PBMC and viral RNA in plasma using a semi-quantitative limiting dilution polymerase chain reaction (PCR). The presence of a syncytium-inducing (SI) phenotype was assessed after direct cocultivation of patient PBMC with MT-2 cells. Results of the quantitative PCR and the MT-2 coculture were correlated with the clinical stage of the disease, with the number of CD4+ T cells, and with the results of other virological and immunological markers, such as the level of p24 antigen, beta 2-microglobulin (beta 2M) and neopterin. RESULTS: Significant differences were observed between the results for asymptomatic and symptomatic patients for all markers under study. In the group of asymptomatic patients with a CD4+ T-cell count > 200 x 10(6)/l, patients with high amounts of proviral DNA had significantly higher amounts of beta 2M, neopterin and viral RNA, they were more frequently p24 antigen-positive and harboured more frequently SI strains than patients with low amounts of proviral DNA. A good correlation between the proviral DNA and the viral RNA levels was observed. Significant changes of viral RNA but not proviral DNA levels were observed after initiation of therapy or when therapy failed. CONCLUSIONS: We demonstrated the relationship between high proviral DNA level in PBMC, high viral load in plasma, elevated beta 2M and neopterin concentrations in serum, and the presence of p24 antigen in serum in a group of asymptomatic patients with a CD4+ T-cell count > 200 x 10(6)/l. We suggest the possible usefulness of proviral load as an early indicator of disease progression. The presence of SI strains is highly correlated with disease; however, SI strains were detected in only 46% of symptomatic patients. It also appeared that the measurement of viral RNA levels is a useful marker for therapy monitoring.

Interruption of reverse transcriptase inhibitors or a switch from reverse transcriptase to protease inhibitors resulted in a fast reappearance of virus strains with a reverse transcriptase inhibitor-sensitive genotype.

OBJECTIVES: To study the effect of the interruption of reverse transriptase inhibitor (RTI) therapy or a switch from RTI to protease inhibitors, on the genotypic drug-resistance pattern of plasma HIV-1. METHODS: Nine patients who completely stopped all medication, and five patients who switched from a treatment with RTI to a regimen containing protease inhibitors only, were studied. Direct sequencing of the plasma HIV-1 RT and protease gene was performed on follow-up samples taken before and after the interruption of treatment. RESULTS: Comparison of the amino acid sequence of the RT and protease genes in successive samples showed the rapid reappearance of wild-type viral variants in 12 of the 14 patients studied. Wild-type virus replaced the mutant strains 14 days to 2 months after the interruption of therapy, even in patients with a long treatment history. CONCLUSION: The results of this study indicate the sustained lower fitness of mutant strains in vivo. As a result, wild-type virus remains capable of outcompeting the RT or protease mutant strains very fast after removal of the drug. These findings highlight the importance of 'treatment history' in addition to genotypic and phenotypic markers determined at one time-point, when making therapeutic decisions for patients.

Exposure to HIV-1 during delivery and mother-to-child transmission.

BACKGROUND: The correlation between the presence of HIV-1 in maternal cervicovaginal secretions and in the infant's oro-pharyngal secretions at birth, and mother-to-child HIV transmission (MTCT) were examined to obtain a better understanding of its mechanism. METHODS: Women without medical and obstetrical complications, living within a reasonable distance of the government hospital in Mombasa, Kenya, were recruited after informed consent. Maternal and infant characteristics were collected. Polymerase chain reaction was used to detect HIV-1 in cervico-vaginal and oro-pharyngal secretions. Infants were tested for HIV-1 by polymerase chain reaction within 48 h and at 6 weeks after delivery. RESULTS: Between April 1998 and April 1999, 228 woman-infant pairs were included in the study. HIV-1 DNA in cervico-vaginal secretions was independently associated with HIV-1 maternal viral load and with infant birth-weight, whereas HIV-1 RNA was associated with maternal viral load and maternal age. HIV-1 DNA in the oropharyngal secretions was also independently associated with maternal viral load. MTCT rate at the age of 6 weeks was 23.6%. Intrapartum and early postpartum HIV transmission was independently associated with maternal viral load [adjusted odds ratio (OR), 1.6; 95% confidence interval (CI),1.0-2.7], detection of HIV-1 RNA in cervico-vaginal secretions (adjusted OR, 3.2; 95% CI, 1.5-7.3) and of HIV-1 DNA in oro-pharyngeal secretions (adjusted OR, 3.2; 95% CI, 1.1-9.0). DISCUSSION: As far as is known, this is the first study showing that infant exposure to HIV-1 in the birth canal and the presence of HIV-infected cells in the infant's oropharyngeal cavity are independently associated with intrapartum and early postpartum MTCT. It supports the hypothesis that MTCT could occur through the oral route.

Haptoglobin polymorphism, iron metabolism and mortality in HIV infection.

BACKGROUND: Three phenotypes of the antioxidant protein haptoglobin are known: Hp 1-1, Hp 2-1 and Hp 2-2. OBJECTIVES: To investigate the outcome of HIV infection according to haptoglobin type. DESIGN AND METHODS: Haptoglobin phenotypes were determined using starch gel electrophoresis in serum obtained from 653 HIV-infected Caucasians in the AIDS reference centers of Gent (n = 184), Antwerp (n = 309), and Luxembourg (n = 160). Survival was compared between haptoglobin types using Kaplan-Meier curves. Plasma HIV-1 RNA was quantified by reverse transcriptase PCR. Serum iron, transferrin saturation, ferritin, and vitamin C were assayed to evaluate iron-driven oxidative stress in 184 HIV-infected patients and 204 controls. RESULTS: The haptoglobin type distribution amongst the patients (17.6% Hp 1-1, 49.9% Hp 2-1, 32.5% Hp 2-2) corresponded to that of the controls. Kaplan-Meier curves showed a higher mortality for the Hp 2-2 group (P = 0.0001; adjusted mortality risk ratio, 1.78; 95% confidence interval, 1.25-2.54). Median survival time was 11.0 years (Hp 1-1 and Hp 2-1) versus 7.33 years (Hp 2-2). Plasma HIV-1 RNA levels prior to antiviral therapy and their increase over 1 year were highest in Hp 2-2 patients (P = 0.03 and 0.003, respectively). The Hp 2-2 type was associated with higher serum iron, transferrin saturation, and ferritin levels and with low vitamin C concentrations. Furthermore, ferritin concentrations were higher in HIV-infected patients than in controls (P < 0.0001). CONCLUSION: HIV-infected patients carrying the Hp 2-2 phenotype show a worse prognosis, which is reflected by a more rapid rate of viral replication (in the absence of antiviral treatment). They also accumulate more iron and oxidize more vitamin C, suggesting that less efficient protection against haemoglobin/iron-driven oxidative stress may be a direct mechanism for stimulating viral replication.

Influence of betaxolol on renal function and atrial natriuretic peptide in essential hypertension.

The hypotensive action of beta-adrenoreceptor blockers is not fully understood, there being a lack of studies focusing on possible relationships between beta-blockers and the secretion of atrial natriuretic peptide (ANP). In 10 patients with essential hypertension, we investigated the influence of betaxolol, a selective beta 1-adrenergic blocking agent, on renal function and on plasma levels of ANP during exercise, volume depletion and volume expansion. Chronic therapy with betaxolol (mean 14.5 mg/day) did not alter glomerular filtration rate and renal blood flow although blood pressure was reduced. Renal vascular resistance decreased from 12795 +/- 1064 dyn/s per cm5 to 10614 +/- 833 dyn/s per cm5 (P less than 0.005). Under betaxolol, basal ANP levels increased from 39 +/- 10 pg/ml to 80 +/- 19pg/ml (P less than 0.01). ANP increased during exercise and volume expansion but was decreased during volume depletion. ANP values observed under betaxolol treatment showed significantly higher values while preserving their dynamic features. We believe that the stimulating effect of betaxolol on ANP may at least partly account for its hypotensive action.

Detection of IL-2 receptor positive tonsillar lymphocytes by monoclonal antibody and protein A coated erythrocytes.

A sensitive rosette method combining staphylococcal protein A coated rabbit red blood cells and the monoclonal antibody anti-Tac was used to search for the presence of interleukin 2 (IL-2) receptor-bearing T lymphocytes in tonsils from patients with chronic tonsillitis. This method revealed the presence of Tac positive T lymphocytes in the tonsils whereas a conventional immunofluorescence technique was unable to do so. To demonstrate that Tac rosette formation resulted in a selective enrichment of IL-2 receptor-bearing T cells, we measured the proliferative response of these cells to exogenous IL-2. The response of Tac rosette positive cells to recombinant IL-2 was always higher than that of the Tac rosette negative or unselected cells, indicating that this rosette method specifically selects T cells expressing IL-2 receptor.

Interleukin-2 stimulated T cell receptor V gamma 3 positive thymocytes do not migrate to the skin.

T cell receptor (TcR) V gamma 3+ thymocytes, which only develop in the fetal thymus, migrate to the skin. IL-2 stimulation of fetal day 18 murine thymocytes results in a cell population of which 45% of the cells express the TcR V gamma 3. In this study, we describe that those IL-2 cultured TcR V gamma 3+ thymocytes have the killing capacity of lymphokine activated killer cells: NK-susceptible as well as NK-resistant tumor cell lines were killed in an MHC-unrestricted manner. Because of these findings, IL-2-expanded TcR V gamma 3+ thymocytes could have a potential use in adoptive immunotherapy for skin-located tumors. Therefore, we analyzed the migration pattern of IL-2-cultured TcR V gamma 3+ thymocytes upon i.v. injection. We describe their initial entrapment in the lungs and subsequent accumulation in the liver. Localization in the skin was practically absent, and did not differ from that of IL-2 cultured adult thymocytes (mainly TcR alpha beta +). The migration pattern was identical in adult and newborn normal mice, and in adult nude mice. Analysis of the expression of asialo-GM1 revealed that it increased strongly after IL-2 culture. The relevance of this change in asialo-GM1 expression with reference to the migration upon i.v. injection is discussed. This study indicates that an improved understanding of the determinants of in vivo localization of IL-2 cultured cells may lead to improved strategies for adoptive immunotherapy of cancer.

LDH isoenzyme distribution in human T gamma lymphocytes.

The lactate dehydrogenase (LDH) isoenzyme distribution was measured in T gamma+ lymphocytes from normal individuals. T gamma+ lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The B:A subunit ratio was clearly lower in the T gamma+ lymphocytes. Phenotyping of the T gamma+ lymphocytes showed a vast majority of OKT11+, OKM1+, OKT3- lymphocytes. In one experiment, T gamma+ lymphocytes were enriched by OKT3 depletion of T lymphocytes. The low B:A ratio was also found in these cells indicating that the LDH pattern is not the consequence of an in vitro activation by immune complexes. As the T gamma+ lymphocytes were considerably enriched in cells having the characteristics of NK cells according to their phenotyping, morphology and NK cell activity, we may assume that NK cells have a low B:A ratio.

Peritoneal dialysis fluids with a physiologic pH based on either lactate or bicarbonate buffer-effects on human mesothelial cells.

Conventional lactate (Lac)-buffered peritoneal dialysis (PD) solutions have turned out to be detrimental to human peritoneal cells, especially because of a low pH. In the present study, we focus on potential differences between Lac and bicarbonate (Bic) as a buffer when adjusted to a physiological pH. All test fluids were buffered with either 40 mmol/L of Lac or 34 mmol/L of Bic, sterile filtered, and adjusted to a pH of 7.4. Osmotic agents used were 1.36% glucose (Glu), 3.86% Glu, 1% amino acids (AA), and 7.5% Glu polymer (Glupoly). Human peritoneal mesothelial cells (HPMCs) were isolated from the omentum majus, grown to confluence, and incubated after the second passage for 15 minutes (37 degrees C and 5% carbon dioxide) with the test fluids. Cytotoxicity was controlled by measuring apoptotic and necrotic cells with cytofluorometry. Aerobic cell metabolism (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assay) and intracellular adenosine triphosphate (ATP) concentrations were measured to assess cell viability. Release of interleukin-6 (IL-6) from HPMCs was determined as a parameter of cellular host defense. No significant difference in apoptosis or necrosis rates was found between the solutions adjusted to normal pH. However, in the MTT assay, Bic solutions were superior to corresponding Lac pendants at an identical pH of 7.4 (P < 0.01). Intracellular ATP concentrations reflected a very similar pattern (P < 0.05). Glupoly in combination with Lac showed an impaired pattern with both the MTT and ATP assays. Regarding IL-1beta-stimulated IL-6 release, there was a small, but not significantly better, response for Bic. Differences in manifest cell cytotoxicity reflected by apoptosis and necrosis rates could not be detected comparing PD solutions buffered with Lac or Bic at a physiological pH. However, distinct parameters of cell metabolism were superior with Bic compared with Lac. Especially Glupoly was inferior in combination with Lac as a buffer.