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Calf satellite I DNA digested with EcoRI and PstI gives three fragments 643, 621 and 83 bp long. Two of them, the 643 and 621 bp were cloned using pBR 322 vector and analyzed by means of the MspI and HaeIII restriction enzymes.
We have identified an H-2K(d)-binding peptide, HER2p780 (PYVSRLLGI), derived from murine HER2/neu (HER2), that can induce HER2-specific murine cytotoxic T lymphocytes (CTL). Weekly vaccination of BALB/c mice by syngeneic dendritic cells pulsed with HER2p780 peptide, entirely common to murine and human HER2, suppressed growth of pretransplanted HER2-expressing syngeneic tumors. A HER2-expressing human cancer cell line SKOV3 transfected with murine H-2K(d) cDNA could also be lysed by HER2p780-specific murine CTLs, indicating that human HER2-expressing cancer cells can process and present the cognate peptide in the context of H-2K(d). Since H-2K(d) and HLA-A2402 molecules have similar anchor motifs, the possibility of inducing HER2-specific CTL activity with HER2p780 in HLA-A2402 individuals was examined. CD8(+) CTL clones specific for HER2-expressing cancer cell lines were established from peripheral blood lymphocytes with HLA-A2402 by repeatedly sensitizing with peptide-pulsed autologous dendritic cells as well as peripheral blood mononuclear cells. Detailed analysis of their specificity revealed that the cytotoxicity of CTL clones is specific for the cognate peptide with HLA-A2402 restriction. The results suggest that HER2p780 is a unique peptide that may function as a tumor rejection antigen peptide in HLA-A2402 individuals, as it was directly proven here to function in a murine tumor system.
A mouse HER2-derived peptide, HER2p63 (A) (TYLPANASL), can induce K(d)-restricted mouse cytotoxic T lymphocytes (CTL) and also function as a tumor rejection antigen in an in vivo assay. Since the anchor motif of mouse K(d) for peptide binding has much similarity to that of human HLA-A2402, we asked if human HER2p63 (T) (TYLPTNASL) could induce HER2-specific CTL in HLA-A2402-positive individuals. Peripheral blood mononuclear cells (PBMC) of HLA-A2402-positive individuals were sensitized in vitro with HER2p63-pulsed autologous dendritic cells prepared from PBMC. CTL clone derived from these specifically lysed HER2-expressing cell lines bearing HLA-A2402. Cytotoxic activity of the CTL clone against the HER2-expressing cell line bearing HLA-A2402 was blocked by antibodies against CD3, CD8, HLA-A24 or MHC class I, and was also inhibited by the addition of excess HER2p63-pulsed C1R bearing HLA-A2402. Killer cells were generated from PBMC of seven healthy individuals and five ovarian cancer patients, all of HLA-A2402 type, by in vitro sensitization with HER2p63-pulsed autologous antigen presenting cells. These killer cells selectively lysed HER2-expressing SKOV3 transfected with HLA-A2402 cDNA, indicating high immunogenicity of HER2p63 in all 12 individuals examined.
Primary as well as secondary proliferative and cytotoxic responses to 2,4,6-trinitrophenyl (TNP)-modified autologous human cells have been studied. Proliferative responses have been obtained both by primary (peak on day 6) and secondary (peak on day 2--3) stimulation. Both responders and nonresponders were found among the panel of unrelated individuals tested. All responders in a secondary reaction also gave significant primary responses. Intrafamilial studies showed that the ability to restimulate a proliferative response followed the major histocompatibility complex haplotype of the responder; in some cases, the two haplotypes differed in their ability to restimulate. Using unrelated individuals typed for HLA-A, B and C, as well as HLA-D and DR, proliferation was shown to occur only when the unrelated stimulator shared HLA-D region products with the responder. In contrast, no HLA restriction was found in cell-mediated lympholysis (CML) (neither in primary nor in secondary responses) in most cases. The data suggest that the observed killing is independent of sensitization. Both responders and nonresponders in proliferation yielded high levels of lysis; no increase of lysis was found in kinetic studies; most allogeneic CML combinations were highly lytic for the TNP-modified responder cells at a time when the lysis of the specific allogeneic target is negligible. These preliminary data suggest that the killing observed might be different from classical T cell-mediated lympholysis.
To investigate the histocompatibility requirements for the macrophage-T cell interaction in the secondary antibody response, splenic T cells from antigen (carrier)-primed F1 hybrid mice were restimulated in vitro with carrier-pulsed F1, parental or allogeneic macrophages. Surviving T cell were cocultured with hapten-primed F1 or parental "B cells" and restimulated with the appropriate hapten-carrier conjugate. The IgG antibody-forming cell response was then measured using a plaque assay. Mapping of the genetic restriction was performed by use of different strain combinations. T helper cells could be restimulated in the presence of macrophages only provided they shared the I-A subregion of the major histocompatibility complex with the F1 T cells frm F1 hybrids restimulated with parental or I-A-identical macrophages were shown to only cooperate with parental B cells of the same I-A haplotype as the macrophages used for restimulation. The defect was at the level of the macrophage, as addition of macrophages of the I-A haplotype used for the restimulation culture reconstituted ability of F1 helper cells to cooperate with the I-A-nonidentical B cells.
OBJECTIVE: To compare maternal cardiac function in women with intrauterine growth restriction (IUGR) to those with small-for-gestational age (SGA) pregnancies (non-IUGR). METHODS: This was a cross-sectional study involving maternal echocardiography and uterine, umbilical and fetal middle cerebral artery Doppler assessment in 52 normotensive women at 20-36 weeks' gestation with SGA fetuses (26 IUGR and 26 non-IUGR). RESULTS: In the IUGR (compared to the non-IUGR) group, maternal cardiac output (CO) was lower (4.7 vs. 6.1 L/min, P<0.001) and total vascular resistance (TVR) was higher (1444 vs. 1088 dynes/s/cm5, P<0.001). The lower CO was due to a lower preload, demonstrated by a reduced stroke volume (59.9 vs. 73.6 mL, P<0.01) and smaller left atrial diameter (LAD) (31.5 vs. 34.1 mm, P=0.01). Mean arterial pressure and diastolic function were similar between the groups. Logistic regression and receiver-operating characteristics curve analysis for detection of IUGR demonstrated that a model using TVR, LAD, fetal middle cerebral artery pulsatility index and gestational age, had a sensitivity of 96.2% and a specificity of 84.6%. CONCLUSIONS: Maternal echocardiography can provide a very sensitive tool for identifying IUGR pregnancies.
Cytotoxic lymphocytes are either MHC-restricted (cytotoxic T-cells) or nonrestricted (natural killer NK-cells), although cells of the monocyte/macrophage lineage are also cytotoxic, and lymphocytes or phagocytic cells expressing Fc-receptors for immunoglobulin can function as antibody-dependent killer cells (referred to as antibody-dependent cellular cytotoxicity: ADCC). Antigen-specific T-lymphocytes recognise their target antigen in the context of MHC class I components, focusing their attack only against those cells expressing the relevant antigen specificity on their cell surface. A more primitive and alternative mechanism exists whereby NK-cells, classified as large granular lymphocytes (LGL), are able to kill in a non-specific manner, not requiring prior sensitisation to antigen. Both antigen-specific T-cells and LGL mediate their cytotoxicity through the release of cytotoxic molecules at the target-effector cell interface. LGL also have a regulatory role in the immune system through the release of cytokines, and can be distinguished from T-lymphocytes by the expression of distinct phenotypic markers (CD16+, CD56+) and they lack CD3 antigen expression or rearranged alpha/beta or gamma/delta T-cell receptor gene products. Cytotoxic activity is positively regulated by interleukin-2 (IL-2) and interferon (IFN), whilst prostaglandins and transforming growth factor-beta (TGF beta) diminish activation and effector pathways. Cytotoxicity mediated by NK- and T-cell populations are principally involved in the defence against microbial infections and neoplasia; the abrogation of cytotoxicity either by direct interaction of 'suppressor factors' with effector cells, or indirectly by reducing cytokine production can inevitably lead to the proliferation of the disease.
BACKGROUND: Anorexia nervosa is an often chronic disorder with high morbidity and mortality. Many people relapse after weight restoration. This study was designed to determine whether a selective serotonin reuptake inhibitor would improve outcome and reduce relapse after weight restoration by contributing to maintenance of a healthy normal weight and a reduction of symptoms. METHODS: We administered a double-blind placebo-controlled trial of fluoxetine to 35 patients with restricting-type anorexia nervosa. Anorexics were randomly assigned to fluoxetine (n = 16) or a placebo (n = 19) after inpatient weight gain and then were observed as outpatients for 1 year. RESULTS: Ten of 16 (63%) subjects remained on fluoxetine for a year, whereas only three of 19 (16%) remained on the placebo for a year (p =.006). Those subjects remaining on fluoxetine for a year had reduced relapse as determined by a significant increase in weight and reduction in symptoms. CONCLUSIONS: This study offers preliminary evidence that fluoxetine may be useful in improving outcome and preventing relapse of patients with anorexia nervosa after weight restoration.
It is known that development of the interhemispheric pathway is accompanied by a dramatic reduction in the number of callosal fibers. By injecting neuroanatomical tracers into the cortex of newborn and adult rats, we examined the distribution of fibers within the corpus callosum both before and after this loss takes place. We found that in both newborn and mature rats, axons from restricted regions of the cortex are grouped into relatively tight bundles within the corpus callosum and that the location of these bundles depends upon the region of cortex from which they originate. These results indicate that callosal fibers that are destined to disappear are not distributed diffusely throughout the corpus callosum of the neonate, but rather that they traverse this commissure through the sector that is appropriate for the area of the cortex from which they originate. This finding suggests that loss of axons does not play a significant role in establishing the overall topographic pattern of the fibers within the callosal commissure.
Exploiting the graft-versus-leukemia (GVL) effect in mismatched transplants requires its separation from graft-versus-host disease (GVHD). We generated leukemia-specific cytotoxic T lymphocytes (CTL) in three haplotype-mismatched, two class I-mismatched and two single HLA-A locus-matched stimulator-responder pairs. Six patients with chronic myelogenous leukemia and one patient with acute myeloid leukemia transformed from MDS were studied. CTL generated after 10 days stimulation with unselected leukemic peripheral blood mononuclear cells inhibited leukemic CFU-GM colony growth (>85% at 10:1 effector:target ratio) with no third-party colony inhibition. In five pairs, responders were cultured separately with leukemia cells, PHA-B or LCL from the stimulator. After 2-4 restimulations, the T cell repertoire was examined by flow analysis using Vbeta-specific antibodies. Test cultures (but not controls) showed preferential expansion of 1-4 Vbeta families either common to two or more stimulators or unique to a particular stimulator. Notably, we elicited leukemia-specific TCR Vbeta expansions on four out of five occasions. In two pairs, responder cells selected for the appropriate leukemia-specific Vbeta family were shown to have leukemia-specific cytotoxicity. These leukemia-restricted T-cells were CD8+ or CD4+ and CD25+ or CD57+. The results support the development of strategies to selectively deplete GVHD and conserve GVL reactivity in mismatched transplants.
The objective of this study was to compare the perinatal outcomes in growth-restricted (IUGR) twins with a matched group of IUGR singletons. We performed a retrospective cohort study evaluating the perinatal outcomes in IUGR twins matched for gestational age at delivery to the next four IUGR singletons (1:4) in our perinatal database. The perinatal outcomes evaluated included rates of uncorrected perinatal mortality (PMR), admission to the neonatal intensive care unit (NICU), respiratory distress syndrome, grades 3 or 4 intraventricular hemorrhage, necrotizing enterocolitis, periventricular leukomalacia, and length of NICU stay. A cohort of twins with IUGR were matched with a cohort of IUGR singletons and compared for perinatal outcomes. A p value < 0.05 was considered significant. During the study period, we identified 99 IUGR twins meeting our inclusion criteria. These were matched with 396 singletons with IUGR. The mean gestational age at delivery for the twins was 34 +/- 3.8 weeks and 35 +/- 3.5 weeks for the singletons ( p = 0.11). The PMR in twins was significantly higher than in singletons (OR, 2.2; 95% confidence interval, 1.1 to 5.7). Perinatal morbidity was similarly significantly higher in IUGR twins. Compared with age-matched singletons, twins with IUGR have higher perinatal mortality and morbidity rates.
Point mutations in somatic cells play a role in the etiology of several classes of human pathologies. Experimental procedures are required that allow the detection and quantitation of such mutations in disease-related genes in tissue biopsy samples without the need for the selection of mutated cells. We describe the genotypic analysis of single base pair mutations in the Taq I endonuclease recognition sequence TCGA, residues 2508-2511 of exon 2 of the human c-H-ras1 gene, by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) approach. The high thermostability of Taq I endonuclease allows the continuous removal of eventual residual wild-type sequences during the thermocycling of the PCR and reduces polymerase errors in the final RFLP/PCR product to a minimum. As few as five copies of a mutant standard containing two base pair changes in the chosen Taq I site could be rescued from 10(8) copies of wild-type DNA. Taq I RFLP/PCR holds promise for the monitoring of mutations in biochemical epidemiology.
Pancreatic beta cells (insulin-producing cells) and neuronal cells share a large number of similarities. Here, we investigate whether the same mechanisms could control the expression of neuronal genes in both neurons and insulin-producing cells. For that purpose, we tested the role of the transcriptional repressor neuron-restrictive silencing factor/repressor element silencing transciption factor (NRSF/REST) in the expression of a battery of neuronal genes in insulin-producing cells. NRSF/REST is a negative regulator of the neuronal fate. It is known to silence neuronal-specific genes in non-neuronal cells. We demonstrate that, as in the case of the neuronal pheochromocytoma cell line PC12, mRNA coding for NRSF/REST is absent from the insulinoma cell line INS-1 and from three other insulin- and glucagon-producing cell lines. NRSF/REST activity is also absent from insulin-producing cell lines. Transient expression of REST in insulin-producing cell lines is sufficient to silence a reporter gene containing a NRSF/REST binding site, demonstrating the role of NRSF/REST in the expression of neuronal markers in insulin-producing cells. Finally, by searching for the expression of NRSF/REST-regulated genes in insulin-producing cells, we increased the list of the genes expressed in both neurons and insulin-producing cells.
BACKGROUND: Current political debate in Sweden is mainly centred on lowering taxes on alcohol in order to "harmonize" prices with those in neighbouring countries, although the evidence of a negative association between prices and alcohol consumption is more than convincing. METHOD: Total per capita consumption figures for twentieth-century Denmark and Sweden are utilized to illustrate the astonishing effects on consumption patterns of active government policies to restrict availability.
To study the histocompatibility restriction between macrophages and helper T cells, carrier primed guinea pig T cells were positively selected in vitro with antigenpulsed macrophages for 7 days and the selected T cells were then mixed with hapten-primed B cells and stimulated with antigen in a modified Mishell-Dutton system. Helper T cells could only be selected with syngeneic, but not allogeneic, antigen-pulsed macrophages and would then collaborate only with syngeneic, but not allogeneic, hapten-primed spleen cells. When F1 T cells were selected with antigen-pulsed parental macrophages they would only collaborate with B cells of the same parental strain as the macrophages used in the selection culture. These results are strongly in support of the view that the primed T cell is activated by carrier determinants of the nominal antigen in association with Ia antigens on macrophages and the helper T cell, in turn, activates B cells which bear the same Ia antigens and determinants of the nominal antigen bound to immunoglobulin receptors on their surface. In addition, in experiments with antigens the response to which is controlled by I-linked genes, we demonstrated that primed (responder X nonresponder)F1 T cells would only collaborate with B cells of the responder parent. The defect appeared to be at the level of the B cell in that the addition to the cultures of antigen-presenting cells of the responder type did not restore the ability of F1 T cells to collaborate with non-responder B cells.
The genetic basis of the restriction imposed on T cell mediating acquired antimicrobial resistance and delayed-type hypersensitivity (DTH) to Listeria in the rat was investigated. Sharing of MHC-coded genes between donors of sensitized T cells and antigen-stimulated recipients was both necessary and sufficient for efficient transfer of both resistance and DTH. Evidence to support this assumption was derived from experiments involving allogeneic transfers within major histocompatibility complex (MHC)-compatible strains and across MHC-barriers. Further support came from linkage studies with backcrossed rats and with the progeny of F1 rats mated with an unrelated strain. An unexpected difference in the compatibility requirements for effective transfer of DTH and resistance was noted in experiments involving the BI strain (formerly called B3). Thus, while B-region compatibility was obligatory for expression of DTH in recipients of sensitized T cells, considerable levels of protection could be transferred to either A-region or B-region compatible hosts.
The quaternary ammonium ions, choline, hexamethonium, and tetraethylammonium, partially reliev the restricted replication of mengovirus in Madin-Darby bovine kidney cells.
We previously observed an increased sympathetic nervous system (SNS) activity that was partly responsible for a defect in the insulin secretion response to glucose after postweaning protein-energy restriction (PER) in female rats. These results, together with other data on low-protein feeding, suggested that a low protein-to-energy ratio (P/E) in the diet could stimulate energy expenditure (EE), but direct measurements of EE have never been reported under conditions of PER. The goal of the present study was thus to quantify the changes induced by PER to body composition, the various parameters of EE, and plasma triiodothyronine levels. PER induced severe growth retardation, but the subcutaneous white and interscapular brown adipose tissue masses were preserved. Basal metabolism, meal-induced thermogenesis, and triiodothyronine levels were increased, but substrate utilization by the working muscles was unaffected. Meal-induced thermogenesis was increased by spontaneous activity in PER rats only. These results suggest that rats adapt to a low P/E in the diet by burning part of their excess nonprotein energy and storing the remaining excess in subcutaneous adipose tissue.