Effect of mycophenolate mofetil regimen on peripheral blood lymphocyte subsets in kidney transplant recipients.

BACKGROUND AND AIMS: There is growing evidence of the effects of immunosuppressive agents on "immune targets" in renal transplantation. Immunological monitoring could indirectly measure the suppressive effect of these drugs and guide early preventive interventions in transplant recipients. Due to the selective antiproliferative effect of mycophenolate mofetil (MMF) on lymphocytes, our goal was to determine whether MMF modulates peripheral blood lymphocyte subsets (PBLS) in kidney allograft patients. METHODS: We assessed absolute CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD19(+), CD16(+)CD3(-) PBLS counts and CD4/CD8 ratios for 12 months in three groups of kidney allograft patients stratified according to maintenance immunosuppressive regimen: group A (n = 31), which started MMF with prednisone (P) + cyclosporine A (CyA), and two control groups, B (n = 19) and C (n = 15) on P + CyA + azathioprine (Aza) and P + CyA regimens, respectively. We compared intra- and intergroup lymphocyte counts and ratios. RESULTS: Intergroup comparisons showed a significant reduction in all PBLS in group A (CD19(+) from 3 months and other subsets from 6 months), whereas there were no significant changes in PBLS in the other group analyses or comparisons. CONCLUSIONS: Our findings suggest that (1) MMF modulates all PBLS in kidney allograft patients, causing a progressive reduction occurring earlier in CD19(+), and (2) we can rule out that these changes were caused by the "natural immunological evolution" of the transplantation. These results could offer a new method for immunological monitoring of transplant patients.

Validity of the hospital discharge diagnosis in epidemiologic studies of biliopancreatic pathology. PANKRAS II Study Group.

BACKGROUND: The aim was to analyse the magnitude, direction and predictors of change in the main hospital discharge diagnosis (HDD) after a clinical expert review, among patients included in a multicentre molecular epidemiologic study of biliopancreatic diseases. METHODS: A total of 602 patients with a suspicion diagnosis of pancreas cancer (PC), cancer of the extrahepatic biliary system (CEBS) or benign biliopancreatic pathologies (BPP) were prospectively recruited at five general hospitals. A structured form was used to collect information from medical records. A panel of experts revised all diagnostic information and established the main clinicopathological diagnosis (CPD) by consensus. RESULTS: Of the 204 cases with a HDD of PC, 176 (86%) were deemed to have a CPD of PC, eight of CEBS, twelve a neoplasm of different origin, four BPP and four syndromic diagnoses. Thus, 28 cases (14%) were false positives. Of the 129 patients with a HDD of CEBS, 15 (12%) were false positives. Nine of the 396 cases with a HDD of non-PC (2%) had a CPD of PC (false negatives), whilst 14 of 471 patients with a HDD of non-CEBS (3%) were deemed to have CEBS. Overall, sensitivity and specificity of HDD for PC were, respectively, 95 and 93%, and for CEBS, 89 and 97%. Cytohistological confirmation and laparotomy were independent predictors of diagnostic change. CONCLUSIONS: Validity of the HDD was high, but its association with some clinical variables suggests that sole reliance on HDD can significantly bias results, and highlights the need to review all HDDs. Alternatively, only patients at high risk of misdiagnosis could be reviewed: primarily, those lacking a cytohistological diagnosis or a laparotomy. No exclusions appear warranted solely on the basis of age, gender or tumour spread.

Weaning induces an increase in the number of specific cytokine-secreting intestinal lymphocytes in mice.

Intestinal immunity differs from systemic immunity in several aspects and is frequently studied separately. In this work we have analysed the frequency of mononuclear cells spontaneously secreting the cytokines IL-2, IL-4, IL-5, IL-6, IL-10, interferon gamma (IFN-gamma) and tumour necrosis factor (TNF-alpha), in Peyer's patches and lamina propria of small intestine in mice by enzyme linked immunosorbent spot (ELISPOT) during 1 month after weaning. We have found a high percentage of spontaneous Th(1)as well as Th(2)cytokine-secreting lymphocytes in both populations, Peyer's patches and lamina propria. An increase in the number of the lymphocytes secreting most of the studied cytokines, at 1 and 2 weeks after weaning, was also observed. These results suggest that the increase in the number of cytokine secreting lymphocytes may be one of the potential mechanisms involved in the development of the intestinal immune system at weaning.

Learning from case reports: diagnostic issues in an epidemiologic study of pancreatic cancer.

Epidemiologic studies on exocrine pancreatic cancer show a large heterogeneity in diagnostic criteria applied to define "caseness." Reanalyses conducted after review of diagnostic information have yielded substantially different results than those based on more crude classifications of disease. During a multicenter prospective study on mutations in the K-ras gene in pancreatic and biliary diseases, hospital diagnoses from 602 patients were reviewed by a panel of experts. There were two main motivations to do so: a generic interest for the quality of the diagnostic data, and the anticipation that a firm diagnosis could be needed to assess whether patients whose tumors did not harbor the mutation were true negatives or false negatives. In addition, the review of diagnoses was helpful to minimize tissue misclassification, and it had a high educational value for clinicians and epidemiologists. This article illustrates why and how this was so through a brief presentation of the 10 most significant cases. With respect to selection and classification of subjects, the main issues that studies on pancreatic cancer need to address are the differential diagnosis of exocrine pancreatic cancer and pancreatitis, the differential diagnosis of exocrine pancreatic cancer and other abdominal tumors, and the use of survival as a hallmark of pancreatic cancer. In epidemiologic studies of pancreatic cancer, it is warranted that a panel of experts centrally reviews all the existing diagnostic evidence (cytohistological and other) of all patients, regardless of whether they have cytohistological confirmation and of their hospital discharge diagnosis.

Cultured human decidual stromal cells express antigens associated with hematopoietic cells.

Although decidual stromal cells (DSC) have classically been considered to play a nutritional role during pregnancy, several reports have demonstrated that they can also exert different immune activities. Furthermore, some authors have occasionally found antigens on DSC normally expressed by immune cells. In this study, we isolated and cultured 12 human DSC lines and studied them with immunocytochemistry and flow cytometry using monoclonal antibodies against antigens associated with hematopoietic cells. Decidual stromal cells exhibited a constant phenotype: they were CALLA (CD10)-positive and DR-positive, although the expression of CD45, the leukocyte common antigen, was found to be very weak or negative. We also detected myelomonocytic antigens CD11b (CR3), CD13, CD16 (Fc gamma RIII) and CD36, although DSC lacked CD14, CD15 and CD33. B cell antigens CD20, CD21 (CR3), CD23 (Fc epsilon RII) and CD24 were expressed. DRC-1, an antigen detected on follicular dendritic cells (FDC), was also observed on DSC. When these cells were cultured in the presence of progesterone, they expressed desmin and prolactin (PRL), findings that confirmed their identity as DSC. The phenotype described, together with the immune activities reportedly carried out by DSC, suggest that DSC may play a role in the maternal-fetal immune relationship.

Routine HLA DRB/DQB oligonucleotide typing by a non-radioactive dot-blot micromethod.

In the last few years the clinical need for HLA genotyping has become evident. However, the routine use of PCR-based DNA typing techniques has been hampered by economical and/or technical considerations. The classical PCR-SSO (product-dot) method has been widely tested and proven to be useful for large-scale HLA DNA typing. However, it is not a suitable method for routine typing of single samples because it takes several days. Using primers and probes for sequences identical to those compiled by the Eleventh International Histocompatibility Workshop, we designed a non-radioactive dot-blot technique in which each hybridization reaction is performed in a microtiter plate well containing PCR-amplified DNA that has been previously dotted on a small nylon membrane, so that a large number of oligonucleotide probes tailed with biotin-14-dATP can be simultaneously tested against the same sample. We studied 23 B-lymphoblastoid cell lines of known HLA genotype to test the method and, so far, it has been validated on more than 100 patients and healthy relatives typed prospectively. This simple, rapid, inexpensive PCR-SSO dot-blot micromethod makes DRB/DQB DNA typing of single samples possible in a short period of time, and is therefore an attractive alternative to serological typing in routine medical practice.

Study of the enzymatic activity of GGT, LDH, PAP and PSA in semen stains: application to age calculation.

The conditions under which semen stains are stored can markedly affect the stability of some of their biochemical parameters. Because of the resistance of semen to processes of degradation and denaturation, the age of the stain may, under optimum conditions, be calculated. We studied six series of semen stain placed on absorbent natural cloth under three different storage temperatures: 5 degrees C (refrigerator), 18-25 degrees C (room temperature), and 38 degrees C (incubator). Seminal fluid was obtained from nonvasectomized and vasectomized individuals aged 25-40 years (mean age 30 +/- 0.32 years, SD 3.06). A total of 240 strains were divided into groups depending on the duration of storage: 24, 48 or 72 h, 1 week, 1, 2, 4 or 6 months. Eluates were analyzed for gamma glutamyltransferase (GGT), lactate dehydrogenase (LDH), prostatic acid phosphatase (PAP), and p30 or prostate specific antigen (PSA). The results of regression analyses showed that a large proportion of the dependent variable (age of the stain) was explained by the rest of the biochemical markers tested. Calculation of the age of the semen stain thus requires consideration of several biochemical parameters.

Comparison of the proportions of leukocytes in early and term human decidua.

The percentages of cells expressing immune markers were determined with immunohistochemistry and flow cytometry in early and term human decidua. Although we found no variation in the proportion of cells of bone marrow origin (CD45+), the percentages of T cells and CD16+ lymphocytes were significantly higher in term decidua. On the contrary, CD56+ lymphocytes, the most abundant leukocyte type in early decidua, decreased at term. These variations may reflex the immunological adaptations of decidua during pregnancy.

Expression of class II HLA molecules by endothelial cells of human decidua.

Many authors have documented a high level of expression of class II HLA molecules by decidua. Although macrophages appear to be responsible for this, we show in this article that endothelial cells (EC) of the venules and capillaries of human decidua also strongly express class II molecules, whereas EC of chorionic villi do not. We discuss this finding in the context of the maternal-fetal immune interaction.

Detection of immunoregulatory lipid-like factors in human amniotic fluid.

The immunosuppressive activity of amniotic fluid (AF) is extensively documented in the mouse. Although this property is due in part to the presence of alpha-fetoprotein (alpha-FP), other immunosuppressive factors are suspected. In this article, we demonstrate that human amniotic fluid lipid extract (AFLE) is inhibitory of, although not cytotoxic to, PHA-activated human lymphocytes, of mouse bone marrow cells, and of different established cell lines of human and mouse source. This effect is shown to be reversible. Under preparative thin layer chromatography (TLC) using chloroform:methanol:water (60:38:8) as solvents, the activity of AFLE migrates to two peaks of inhibition with Rf values of 0.46-0.62 and 0.84-1, respectively. These lipid-like factors may play a role as a nonspecific immunoregulatory mechanism which prevents maternally mediated immune rejection of the conceptus.

Short-lived suppressor cell activity during normal human pregnancy.

Short-lived suppressor cell (SLSC) activity was determined in normal pregnant women. This activity was significantly increased in all three trimesters of pregnancy and during the first week postpartum. When pregnant women were divided into primiparous and multiparous groups, no significant differences were found between the two groups in any of the periods studied. These results suggest that increased SLSC activity may play a role in the materno-fetal tolerance and that parity has no influence on this activity.

Decreased levels of circulating CD4+ T lymphocytes during normal human pregnancy.

Peripheral blood mononuclear cell (PBMC) populations during human pregnancy have been investigated by many authors, although the different results obtained, principally in relation to T cells, are very discrepant. In this study we aimed to exclude all the possible causes of these discrepancies: small sample size; diurnal rhythm of CD4+ T cells; smoking habits; haemodilution which occurs during pregnancy and inappropriate statistical analysis; in order to determine whether gestation has a definite effect on PBMC populations. We found that the percentage of CD4+ T lymphocytes decreases in the first and second trimesters, returns to the non-pregnant level in the third trimester and remains there in the postpartum period. The percentages of CD3+ T lymphocytes run parallel to those of CD4+ while CD8+ T lymphocytes do not vary. The proportion of CD16+ cells, which include mature NK cells, diminishes in the second trimester and this reduction is maintained in the third trimester and the puerperium. No variation was found in the other PBMC studied (CD20+ lymphocytes, CD14+ monocytes and D/DR+ cells). When parity was considered no difference was seen between primiparous and multiparous women in any of the cell populations tested.

Presence of Langerhans cells in the cornea of Klebsiella keratoconjunctivitis mice.

Frozen sections of normal Balb/c corneas and corneas from Balb/c mice with Klebsiella keratoconjunctivitis were examined for the expression of class I, class II H-2 antigens and MAC-1 antigens using monoclonal antibodies in an immunoperoxidase technique. Class I antigens were readily detected, in both normal and diseased corneas, mainly in the epithelium. Class II (Ia) and MAC-1 antigens were not detected in the normal corneas. However, these two antigens were found mainly in the epithelium and to a lesser extent in the stroma of corneas from keratoconjunctivitis mice. Both normal and diseased corneas were furthermore shown to be peroxidase-. Since Langerhans cells (LC) are Ia+, MAC-1+, and peroxidase- cells, we conclude that although the normal mouse cornea is devoid of these cells, under bacterial infection LC infiltrate the corneal epithelium.

Detection in a murine T lymphoma of a lipid-like factor that inhibits cell growth.

In this paper we show that MCG3 cells, a murine T lymphoma, contain a factor(s) that inhibits the proliferation of cells of different histological origin. The lack of sensitivity of this cell proliferation-inhibiting factor (CPIF) to the treatment with proteolytic enzymes and its solubility in organic solvent demonstrated that it is a lipid-like substance. Separation by thin-layer chromatography showed it migrates before the prostaglandins with activity on cell proliferation. CPIF activity was reversible and more intense on bone marrow cells than on tumor cells, suggesting that it can play a role in cell growth regulation.

Controlled trial of endoscopic sclerosis in bleeding peptic ulcers.

Of 113 patients in whom endoscopy revealed a bleeding gastric or duodenal ulcer 55 were randomly allocated to receive endoscopic sclerosis (ES) (injections of adrenaline/polidocanol) plus cimetidine while 58 received cimetidine alone as controls. 3 patients treated with ES (5.5%) compared with 25 controls (43.1%) had a major recurrent haemorrhage during their hospital stay. ES also led to significant reductions in the need for emergency surgery (3 vs 20 patients), transfusion requirements (mean 0.42 [SD 1.1] vs 2.7 (3.19) U), and the length of hospital stay (11.6 [5.1] vs 16.2 [11.3] days). ES as an adjunct to conventional medical treatment is an effective and safe emergency therapy for gastrointestinal bleeding due to peptic ulcer.

Detection of a cell proliferation-inhibiting factor in the cell-free ascitic fluid of intraperitoneal mouse tumors.

Three cell-free ascitic fluids: MCG3AF, P815AF and SL2AF were obtained from mice injected with MCG3, P815 and SL2 tumor cells, respectively. These ascitic fluids were tested in culture with different normal and tumor mouse cells showing an inhibitory effect on 3H-thymidine and 3H-uridine uptake. The MCG3AF, selected for further study, was tested with a larger number of mouse tumor cell lines and also with normal mouse and human cells. This ascitic fluid inhibited 3H-thymidine and 3H-uridine uptake in all cases. The cell proliferation-inhibiting factor(s) (CPIF) detected in MCG3AF was found to be thermostable and nondialyzable. Further biochemical analysis showed that CPIF is a lipid but with no relationship with very low-density lipoproteins.

[Study and characterization of lipid factors that inhibit cellular proliferation in MCG3 rat T lymphoma]. / Estudio y caracterización de factores lipídicos inhibidores de la proliferación celular (FLIPC) en el linfoma T de ratón MCG3.

Lipid factors that inhibit cellular proliferation have been detected in MCG3 cells, a T lymphoma of B10 mouse. The inhibiting effect is cytostatic, non cytotoxic, reversible and lacks species and tissue-specificity. These lipids have been isolated by Thin Layer Chromatography (chloroform:methanol:water, 60:38:8) showing an Rf100 between 36 and 42, similar to that shown by gangliosides and phosphatidylinositol.

Anapsos, an antipsoriatic drug which increases the proportion of suppressor cells in human peripheral blood.

Anapsos, an antipsoriatic drug, was administered to normal volunteers in order to study its possible activity on the immune system. After three days of treatment, the drug decreased the lymphoblastic response to PWM and very slightly reduced the serum levels of immunoglobulins. After five days of treatment, however, both values were normal. The response to ConA decreased, and there was an increase in the suppressor index and in the proportion of OKT 8+. The drug did not vary the proportion of OKT 4+ and OKT 3+ cells. These results suggest that anapsos acts by increasing the number of suppressor cells. Such anapsos-induced suppressor cells are probably responsible for the diminished response to ConA, but did not seem to significantly affect response to PWM and serum levels of Ig after five days of treatment.