Co-localization of neuroendocrine hormones in the human fetal pancreas.

OBJECTIVE AND DESIGN: Co-localization of the four major pancreatic hormones, and also of islet amyloid polypeptide (IAPP), peptide tyrosine tyrosine (PYY), secretin and neurotensin, has been studied in the endocrine pancreas of human fetuses at 16, 18 and 22 weeks of gestation. METHODS: Double and triple immunofluorescence stainings have been used. RESULTS: All three fetal pancreata contained cells that showed insulin, glucagon, somatostatin, pancreatic polypeptide (PP), IAPP, secretin and PYY immunoreactivity. Neurotensin cells were found in the youngest fetus and gastric inhibitory polypeptide (GIP) in the two older fetuses. Co-localization of two hormones occurred in most of the endocrine cell types in the three fetuses examined, but three hormones occurred in only a few cells and especially in the youngest fetus. Somatostatin cells were the only cell type which was largely monohormonal. Our findings showed that there are two different co-localization patterns: insulin was co-localized mainly with IAPP and glucagon, while secretin and PYY occurred together with glucagon and PP. CONCLUSIONS: These data are the first to describe secretin and neurotensin in the fetal pancreas. Two different co-localization patterns could be distinguished: insulin, IAPP and glucagon, and glucagon, secretin, PP and PYY.

p-Aminobenzoic acid, but not its metabolite p-acetamidobenzoic acid, inhibits thrombin induced thromboxane formation in human platelets in a non NSAID like manner.

We have previously shown that p-aminobenzoic acid (PABA) is acetylated by several cell lines and most peripheral blood cells, including platelets, to p-acetamidobenzoic acid (PACBA). The structural similarity of PABA and PACBA to local anesthetics and some non steroidal anti inflammatory drugs urged us to perform the present investigation. When human platelets were stimulated with thrombin to liberate AA, we found that PABA inhibited the production of thromboxane (TxB2) as measured with enzyme-linked immunosorbent assay. The inhibition was reversible and observed at PABA concentrations ranging between 55 and 1000 microM. At 328 microM PABA the production of TxB2 diminished by 87% (p = 0.013). PACBA in the same doses did not affect the production of TxB2. When platelets were incubated with [1-14C]AA, in the presence of PABA, the production of [1-14C]TxB2 was only slightly inhibited, according to analysis by high pressure liquid chromatography. Obviously PABA is not mainly acting as a prostaglandin H (cyclooxygenase) or Tx synthase inhibitor. It is rather affecting a step prior to thromboxane production, most likely the liberation of the precursor AA. In conclusion, our results demonstrate for the first time that PABA, a substance occurring in nature, inhibits endogenous TxB2 synthesis in human platelets and might thus exert profound effects on platelet AA metabolism.

Arachidonic acid is a preferred acetyl donor among fatty acids in the acetylation of p-aminobenzoic acid by human lymphoid cells.

We have previously reported that human lymphoid cells, such as peripheral blood mononuclear leukocytes (PBML) and the T-cell leukemia line Jurcat, synthesize p-acetamidobenzoic acid from p-aminobenzoic acid (PABA) and a two carbon fragment from arachidonic acid (AA), conceivably derived from beta-oxidation. Here we demonstrate that AA is a preferred substrate in this acetylation reaction over other common fatty acids such as palmitic (PA), oleic, linoleic or linolenic. This was unexpected because AA is not considered as a fuel fatty acid. In Jurcat cells, AA is also preferred as a substrate for beta-oxidation over PA. In contrast, in PBML, PA was clearly preferred as substrate for beta-oxidation over AA, in accordance with previous observations. The difference between Jurcat cells and PBML was not dependent on culture conditions, because phytohemagglutinin and interleukin-2 activated PBML, kept in culture, showed the same PA preference as freshly prepared non-activated PBML. Furthermore, we observed differences between Jurcat cells and PBML in their relative content of fatty acids and in the incorporation of PA and AA into triacylglycerols and phospholipids. Taken together, our results show differences in beta-oxidation between Jurcat cells and PBML, and suggest the involvement of peroxisomal, besides mitochondrial, beta-oxidation, in the acetylation of PABA with fatty acids as acetyl donors.

Identification of a substance, previously shown to enhance mitogenesis of human lymphocytes, as the acetamide of p-aminobenzoic acid.

We characterize here an arachidonic acid (AA)-derived metabolite previously found to have an adjuvant effect in phytohemagglutinin-induced mitogenesis of lymphocytes from mothers of newborn babies and from immunodeficient infants. We named the metabolite 'compound 4' due to its position in a thin-layer chromatography system developed for isolation of eicosanoids. The compound was originally found to be produced by peripheral blood mononuclear leukocytes and the T cell leukemia line Jurcat after long-term (18-24 h) incubation with [1-14C]AA. Compound 4 is also produced by lymphocytes, monocytes, platelets, thrombocytes, cultured fibroblasts and various types of malignant cell lines. We purified this metabolite by means of high pressure liquid chromatography with synchronous detection of radioactivity and measurement of ultraviolet-light absorption at 278 nm. Proton nuclear magnetic resonance spectroscopy and mass spectrometry with electron impact techniques demonstrated that compound 4 is not an eicosanoid, but is identical to p-acetamidobenzoic acid (PACBA). The cells synthesize PACBA from p-aminobenzoic acid and a two-carbon residue from AA.

Differential sensitivity to cAMP among human cord and maternal/adult peripheral lymphocytes discloses differences between PHA- and OKT3-induced activation pathways.

Previous studies have demonstrated that human cord blood lymphocytes are resistant to the anti-proliferative action of prostaglandin E2 (PGE2) in phytohaemagglutinin (PHA)-induced mitogenesis, whereas the same cells activated by OKT3 are at least as sensitive to PGE2 as the corresponding cells from their mothers or other adults. In the present investigation it was found that: (i) PHA-induced proliferation of cord peripheral blood mononuclear leucocytes (PBML) is less sensitive to inhibition by forskolin--a direct activator of adenylate cyclase (AC)--and dibutyryl cAMP--a permeant cAMP analogue--than the proliferation of the corresponding maternal cells; (ii) OKT3-induced proliferation of cord as well as maternal PBML is highly sensitive to inhibition by forskolin and dibutyryl cAMP; (iii) cord PBML show an overall lower rate of AC activity compared with maternal PBML, in response to PGE2 and other autacoids as well as to receptor-independent stimulation; (iv) cord PBML also display a significantly lower rate of degradation of cAMP through cAMP-phosphodiesterase compared with maternal cells; (v) unbroken cord and maternal PBML show comparable rates of cAMP accumulation after stimulation with PGE2. The results suggest a lower sensitivity to the effect of cAMP in cord compared with maternal/adult lymphocytes in PHA-induced proliferation. Moreover, the data illustrate differences between PHA- and OKT3-mediated activation pathways, as well as differences in cell regulation mechanisms between cord and maternal PBML.

Inflammatory cells in transplanted kidneys are infected by human cytomegalovirus.

To determine which cells in kidney grafts are infected with human cytomegalovirus (HCMV) before and after transplantation, kidney specimens were studied by in situ hybridization with 35S-labeled DNA probes representing HCMV immediate-early and late genes. Pretransplantation biopsies and serial posttransplantation biopsies were obtained from 7 renal grafts. All of the transplant recipients were HCMV-seronegative at the time of transplantation and all developed primary HCMV infections. HCMV nucleic acids were not detected in biopsies taken from the healthy donor kidneys before transplantation. However, biopsies taken at various intervals after transplantation showed abundant hybridization with HCMV immediate-early and late gene probes. Virtually all of the hybridizing cells were mononuclear inflammatory cells in the interstitial spaces of the kidney. Occasional hybridization was seen with renal tubular or glomerular cells. No cytomegalic cells were seen. Biopsy specimens taken after systemic anti-HCMV chemotherapy with phosphonoformate showed no uniform reduction in HCMV gene expression. These studies demonstrate that the principal HCMV-infected cells in kidneys of renal transplant patients with primary HCMV infections are infiltrating inflammatory cells.

A novel arachidonic acid metabolite with stimulatory effect on PHA-induced mitogenesis of lymphocytes.

The isolation of a novel arachidonic acid (Aa) metabolite from the supernatant of unstimulated human cord blood mononuclear leucocytes is reported. The metabolite, arbitrarily named 'compound 4' is neither a known lipoxygenase nor a cyclooxygenase product. 'Compound 4' was added to PHA-stimulated peripheral blood mononuclear leucocytes (PBML) from healthy blood donors, from mothers at term and from patients with immunodeficiency. 'Compound 4' induced an increase in the 3H-TdR incorporation by the maternal PBML and by the PBML from patients with various immunodeficiencies such as Wiskott-Aldrich syndrome and common variable immunodeficiency, whereas it had no effect on the proliferation of PBML from blood donors.

A prostaglandin-mediated suppressive activity of cord as compared to maternal or other adult adherent cells in OKT3 antibody-induced proliferation.

In a previous study we reported that cord blood lymphocytes show lower OKT3 responses as compared to their mothers and to other, unrelated adults. In the study reported here, we investigated the interactions between lymphocytes and adherent accessory cells in OKT3-stimulated cultures of newborn (cord), maternal, and other adult peripheral blood mononuclear leukocytes (PBML) and determined the following. (1) Removal of adherent cells (AC), by two cycles of plastic adherence or by nylon wool columns, impaired the OKT3-induced proliferation of maternal/adult cells, but significantly enhanced the OKT3 responsiveness of cord cells. (2) Addition of indomethacin, and other prostaglandin (PG) synthesis inhibitors, caused a more than twofold augmentation of cord PBML OKT3 responses, but had only a small, if any, enhancing effect on maternal/adult PBML. Cord PBML cultures deprived of AC were no longer enhanced by indomethacin. (3) Exogenous PGE2 (1.4 X 10(-6) through 1.4 X 10(-9) M) strongly inhibited OKT3-induced proliferation of maternal, cord, and adult PBML, at a wide range of antibody concentrations (5-100 ng/ml). However, an obvious difference in the extent of PG-mediated inhibition was observed among these three populations, and the order of PG sensitivity, from most to least sensitive, was cord greater than maternal greater than adult. (4) Purified interleukin-1 (IL-1) could not replace the accessory function of AC in the OKT3-induced proliferation of maternal/adult lymphocytes. In contrast, IL-1 increased by greater than 50% the OKT3 responsiveness of cord PBML in the absence, but not in the presence, of cord monocytes. Our observations strongly argue for a distinct, predominantly suppressive function of cord monocytes as compared to maternal/adult monocytes in OKT3-induced mitogenesis, and indicate prostaglandins as major mediators of this suppression.

Monocyte-regulated hyporesponsiveness of human cord blood lymphocytes to OKT3-monoclonal-antibody-induced mitogenesis.

OKT3 monoclonal antibody recognizes surface antigenic structures present on all human mature T lymphocytes and is mitogenic for resting peripheral T cells. Recent reports suggest that these structures are linked to the specific antigen receptor of the T cells and play an important role in T-cell activation. We have tested the mitogenic action of OKT3 on resting lymphocytes from human newborns, their mothers, and unrelated adults. We found that the proliferative response of cord T cells to OKT3 is significantly lower than the response of maternal and adult cells at all doses of the antibody tested (5-1000 ng/ml). This difference was not dependent on culture conditions (source of serum, kinetics induced by the OKT3 antibody, or different proportions of adherent cells in peripheral blood mononuclear leukocytes), and could only to some extent be accounted for by differences in the proportions of OKT3-binding cells between these populations. Removal of adherent monocytes largely diminished the OKT3-induced proliferation of maternal and adult cells, by an average of 70-80%. In contrast, it significantly enhanced the proliferation of cord cells. The proliferative response of cord T lymphocytes to the two polyclonal T-cell activators phytohaemagglutinin and concanavalin A was similar to or greater than that of mothers and other adults.

Conditions for the immunohistochemical demonstration of complement factor C3 in formaldehyde-fixed and paraffin-embedded renal tissues.

Paraffin sections of formaldehyde-fixed renal biopsies were labeled for complement C3 by a polyclonal rabbit antibody to human complement C3, by the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin peroxidase complex (ABC) techniques, respectively. All tissues had C3 deposits according to direct immunofluorescence on fresh frozen sections. Staining for muramidase was introduced as an intrinsic control for the degree of tissue proteolysis after the necessary trypsin digestion prior to the immunoenzyme labeling. The results indicated that even minute deposits of C3 could be detected in paraffin sections by the ABC method, which was more sensitive than the PAP technique; the ABC method allowed a maximal dilution of 1:2,400 of the primary antibody as compared to 1:800 for the PAP technique.

Human fetal/neonatal suppressor activity: relation between OKT phenotypes and sensitivity to prostaglandin E2 in maternal and neonatal lymphocytes.

T lymphocytes from human fetuses and newborns strongly and spontaneously suppress various adult cell functions (i.e. T-cell proliferation, B-cell differentiation, and Ig synthesis). The precise phenotype of the suppressor cell is controversial. In this investigation we use cord T-cell subsets negatively selected by the panning technique or by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Cord T cells deprived of the OKT4+ subpopulation exerted only a marginal suppressor activity (12 +/- 7 as compared to 73 +/- 4% of unfractionated T cells) on the proliferation of maternal cells in our PHA-stimulated co-culture assay using sex chromosomes as markers for dividing cord (male) and maternal cells. The suppressive effect was direct, i.e. not mediated by induction of maternal OKT8+ suppressor effector cells. Cord and maternal T-cell subsets were also tested for their sensitivity to exogenous prostaglandin E2 (PGE2) at doses varying between 1.4 X 10(5) and 1.4 X 10(9) M. Both maternal OKT4- and OKT8- T-cell subsets were highly sensitive to suppression by PGE2. In contrast, cord OKT8- T cells were essentially nonsensitive at all doses of PGE2 used, whereas cord OKT4- T cells were significantly suppressed at four out of five concentrations tested (1.4 X 10(6) through 1.4 X 10 (9). Our results suggest a direct correlation between the phenotypes of the cord-suppressor and maternal-target T cells and their sensitivity to PGE2.

Blood transfusions generate/increase previously absent/weak blocking antibody in women with habitual abortion.

Women who suffered recurrent spontaneous abortions of unknown cause were studied for cellular reactivity and blocking antibody in a one-way mixed lymphocyte culture. A defined group of 20 women whose serum displayed no blocking capacity was given three transfusions of leukocyte-rich erythrocyte concentrates. Serum from all women displayed significant blocking capacity 2 months after the third transfusion. Because blocking antibody seems to be one of the necessary prerequisites for successful pregnancy, leukocyte transfusions from third-party donors ought to be an effective cure for habitual abortion in selected cases.

Gastrointestinal carcinoma-associated antigen detected by a monoclonal antibody in dysplasia and adenocarcinoma associated with chronic ulcerative colitis.

Colorectal tissue specimens from 13 patients with chronic ulcerative colitis, of whom all had epithelial dysplasia and 2 had adenocarcinoma, were tested for the presence of gastrointestinal carcinoma-associated antigen (GICA), using an immunoperoxidase technique with a monoclonal antibody (MAb) against this antigen. GICA was present in the formaldehyde-fixed and paraffin-embedded sections of dysplastic and cancer tissue but absent from normal or hyperplastic epithelium. However, the pattern and extent of staining with the antibody did not correlate with the degree of dysplasia, i.e., "mild" dysplasia was often positive, and "severe" dysplasia was sometimes negative. Changes classified as "indefinite for dysplasia but probably negative" were variable in their expression of GICA. The adenocarcinomas were selectively labelled within cell clusters. In contrast, ulcerative colitis (UC) patients with severe inflammatory changes but with no detectable dysplasia were negative for GICA. GICA could be eluted from paraffin blocks of dysplastic tissue and biochemically characterized as a glycolipid. The detection of this antigen might be a useful complement to morphological examination in discriminating between precancerous and benign epithelial lesions of the colon.

Strong prostaglandin associated suppression of the proliferation of human maternal lymphocytes by neonatal lymphocytes linked to T versus T cell interactions and differential PGE2 sensitivity.

Lymphocytes from human fetuses and newborns strongly, regularly, and non-specifically suppress the proliferation of PHA stimulated peripheral blood mononuclear leucocytes. The suppression is prostaglandin (PG)-dependent. Our present investigation clearly indicates that the suppression is associated with neonatal T versus maternal T lymphocyte interactions, independent of monocytes. This was borne out from co-cultures of PHA stimulated maternal and male cord T cells enriched by nylon wool columns (greater than 90% T3+ cells; residual adherent cells ranging between 0 and 0.05%, and sIg+ cells between 0.6 and 3.2%). Sex chromosomes served as markers for dividing cord (male) or maternal cells. Each of three separate PG synthetase inhibitors introduced into the co-cultures-indomethacin 28 microM, 5, 8, 11, 14-eicosatetraynoic acid (ETYA) 33 microM, or Naprosyn 217 microM--decreased the suppression of the maternal T cells by a maximum of 65%, indicating the importance of PG for the suppression. Moreover, exogenous PGE2 ranging between 1.4 X 10(-5) and 1.4 X 10(-9) M strongly suppressed the proliferation of PHA stimulated maternal T cells (ranging between 62 and 26%) but left the proliferation of cord T cells virtually unchanged. This difference offers one explanation for the strong and invariable suppression of adult lymphocytes by fetal/neonatal lymphocytes. The suppression might be of importance for prohibiting rejection of the placenta by maternal lymphocytes.

Expression of gastrointestinal carcinoma-associated antigen (GICA) detected in human fetal tissues by monoclonal antibody NS-19-9.

The expression of a gastrointestinal carcinoma-associated antigen (GICA), a monosialoganglioside, was investigated in tissues from human fetuses of various gestational ages (10-40 weeks). Mouse monoclonal antibody NS-19-9, generated in mice immunized with SW1116 human colon carcinoma cells, was used along with a second polyclonal antibody to mouse IgG to detect antigen expression as visualized by means of the biotin-avidin-peroxidase assay. Sections of snap-frozen tissues or tissues fixed in 4% formaldehyde, in mercury chloride-formaldehyde, or in Bouin's solution were used. GICA was consistently detected in the mucosal epithelium of the small intestine. In contrast, the mucosal epithelium of the colon-rectum contained no detectable GICA, nor could the antigen be detected biochemically in extracts from the large intestine. GICA was usually found in the epithelium of the larynx, trachea and main bronchi as well as in the conjunctiva, lacrimal and salivary glands, gall bladder and ductus choledochus, and in the epithelium of the renal pelvis of early and mid-gestation fetuses.

Habitual abortion: parental sharing of HLA antigens, absence of maternal blocking antibody, and suppression of maternal lymphocytes.

The immunologic responsiveness of eight women who habitually abort has been investigated. All shared an HLA-A or B antigen with their husbands. Sharing of an HLA-DR antigen was found in seven couples, one of which also had a second DR antigen in common. The probability for this high frequency of HLA-DR sharing is negligible (p = 0.0004), as calculated from the antigen frequencies among Europeans. Cells from the woman with two shared DR antigens displayed a minor response to her husband's cells but reacted strongly to control cells, whereas the other women's cells reacted normally to cells from both their husbands and controls in one-way mixed lymphocyte culture (MLC). Only minor cytotoxicity was displayed by women's cells in a direct cell-mediated lympholysis (CML) assay, but they mounted normal cytotoxic responses against both husbands' cells and control cells in an amplified CML assay. The sera from six of the habitually aborting women displayed no blocking activity in one-way MLC, and seven of them had no cytotoxic antibodies. Cells from all habitual aborters were suppressed in two-way MLC by cells from husbands and most controls. We hypothesize that increases in HLA compatibility between mother and fetus and in maternal susceptibility to suppressive influences are in some way linked to a deficiency in the development of antifetal antibody during pregnancy. As a consequence, the fetus may be deprived of the protection by maternal blocking antibody, which may allow maternal cytotoxic reactions to cause abortion.

Strong suppression by mononuclear leukocytes from cord blood of human newborns on maternal leukocytes associated with differences in sensitivity to prostaglandin E2.

We have tested peripheral mononuclear leukocytes (PML) from the cord blood of newborns, from sera of their mothers, and from sera of nonrelated nonpregnant adult women for sensitivity to suppressive exogenous prostaglandin E2 (PGE2). Endogenous PG production was simultaneously inhibited by indomethacin 2.8 microM. The phytohemagglutinin-stimulated (PHA-simulated) uptake of tritiated thymidine (3H-TdR) by PML from the mothers and the nonpregnant women was suppressed by the exogenous PGE2 at a concentration of 1.4 x 10(-8) M, 100 times less than the one required to suppress the PML from newborns (1.4 x 10(-6) M). In addition, 1.4 x 10(-7) M or less of PGE2 reversed the suppression of neonatal PML to stimulation. The maternal PML were reversed into stimulation at 1.4 x 10(-9) of exogenous PGE2. The amount of endogenous PGE2 synthesized by 1 x 10(6) fresh, nonstimulated neonatal PML according to gas chromatography-mass spectrometry assay was 5 ng (1.4 x 10(-8) M). The synthesis increased to 27 ng/10(6) cells after 18 hours' incubation. These concentrations are similar to the ones of exogenous PGE2 at which neonatal PML were slightly stimulated but the maternal cells were still suppressed. Preincubation for 18 h at 37 degrees C decreased the PGE2 induced suppression of the adult PML but did not change the response of the neonatal PML.