Genetic alterations in primary and secondary hyperparathyroidism

OBJECTIVE: To evaluate treatment of patients with primary liver cancer. DESIGN: Prospective protocol including subsets of randomised studies. SETTING: University hospital, Sweden. SUBJECTS: 123 patients with primary liver cancer. INTERVENTIONS: 64 patients underwent hepatic resection, 25 were included in a trial of adjuvant chemotherapy. 24 further patients whose tumours were not resectable were included in a trial of intra-arterial infusion of doxorubicin. MAIN OUTCOME MEASURES: Survival and postoperative morbidity. RESULTS: The median survival time for patients who had had resections was 11 months (range 0-111). Twelve per cent survived more than 5 years. No prognostic factor had any significant effect on outcome. The postoperative mortality was 11% (7/64). The patients allocated to adjuvant chemotherapy survived a median of 10 months (range 1-47) and the controls 29 months (range 8-111) (p=0.04). Patients with unresectable liver cancer treated with intra-arterial doxorubicin lived no longer than untreated controls (median 8 months (range 1-56) compared with 7 months (range 1-28)). CONCLUSIONS: Treatment of patients with primary liver cancer is still an unsolved problem. Adjuvant chemotherapy with doxorubicin had no beneficial effect on survival.     

CD4-mediated and CD8-mediated cytotoxic and proliferative immune responses to Toxoplasma gondii in seropositive humans

Both CD4+ and CD8+ cytotoxic T lymphocytes (CTL) are part of the human immune response to Toxoplasma gondii infection. To further our understanding of Toxoplasma immunity, we investigated factors influencing stimulation of CD4+ or CD8+ human T. gondii-specific immune cells. Both antigen-pulsed and Toxoplasma-infected antigen-presenting cells (APC) induced cell proliferation. Toxoplasma-infected APC elicited strong proliferation of CD4+ cells, but little or no proliferation of CD8+ cells, unless high antigen loads were used. Toxoplasma-infected APC stimulated specific cytotoxicity poorly or not at all, owing to death of stimulated cultures, whereas antigen-pulsed APC strongly elicited specific cytotoxicity. Cytotoxicity elicited by either type of APC resided exclusively in CD4+ T cells in polyclonal cultures. Thus, Toxoplasma-infected APC elicited stronger CD4-mediated than CD8-mediated cell proliferation and generated CD4+ CTL more readily than CD8+ CTL. Nonetheless, specific CD8+ memory cells were demonstrated, and rare CD8+ Toxoplasma-specific CTL were subcloned. Fixed Toxoplasma-infected APC (which induce CD8+ CTL) also elicited cell proliferation, but polyclonal cultures stimulated with these infected APC did not die. Unfixed Toxoplasma-infected APC strongly inhibited phytohemagglutinin-induced cell proliferation, whereas fixed APC did not. These data suggested that infected APC were inhibitory or lethal to some immune cells. Further investigations into interactions between immune cells and Toxoplasma-infected cells likely will help elucidate factors involved in the immunopathogenesis of Toxoplasma infection. As other intracellular parasites, including Plasmodium spp. and Leishmania spp., also elicit CD4+ CTL, such work may help establish paradigms governing immunity to intracellular parasites.     

Antigen-specific T-cell responses during primary and secondary Listeria monocytogenes infection

Although murine listeriosis is a widely used experimental model for the analysis of cell-mediated immunity, there is little information about individual T-cell antigens of Listeria monocytogenes which are recognized during primary and secondary infection. To study the antigen responses of L. monocytogenes-reactive T cells, somatic and secreted listerial proteins were separated by two-dimensional gel electrophoresis and subsequently divided into 480 liquid fractions. Antigen-specific T cells isolated from mice at different times of primary and secondary listeriosis were tested for their capacity to proliferate with distinct protein fractions. Supernatants of these cultures were screened for the production of gamma interferon, interleukin-4 (IL-4), and IL-10. Proliferation of antigen-specific T cells correlated with the production of high concentrations of gamma interferon, whereas IL-4 and IL-10 production in response to listerial protein fractions could not be detected. During both primary and secondary listeriosis, T cells recognized a multitude of somatic and secreted proteins rather than one or a few dominant antigens. Secreted proteins were recognized before somatic proteins, and T cells recognized different fractions in secreted and somatic proteins.     

Sensorimotor integration in human primary and secondary somatosensory cortices

We measured somatosensory evoked fields (SEFs) to electric median nerve stimuli from eight healthy subjects with a whole-scalp 122-channel neuromagnetometer in two different conditions: (i) 'rest', with stimuli producing clear tactile sensation without any motor movement, and (ii) 'contraction' with exactly the same stimuli as in 'rest', but with the subjects maintaining sub-maximal isometric contraction in thenar muscles of the stimulated hand. The aim was to study the role of the primary (SI) and secondary somatosensory (SII) cortices in sensorimotor integration. The amplitude of the SI response N20m did not change with coincident isometric contraction, whereas P35m was significantly reduced. On the contrary, activation of contra- and ipsilateral SII cortices was significantly enhanced during the contraction. We suggest that isometric contraction facilitates activation of SII cortices to tactile stimuli, possibly by decreasing inhibition from the SI cortex. The enhanced SII activation may be related to tuning of SII neurons towards relevant tactile input arising from the region of the body where the muscle activation occurs.     

Two distinct populations of primary cytotoxic cells infiltrating into allografted tumor rejection sites: infiltration of macrophages cytotoxic against allografted tumor precedes that of multiple sets of cytotoxic T lymphocytes with distinct specificity to alloantigens

The majority of cases with familial Alzheimer's disease (FAD) are linked to mutations of the presenilin (PS) genes. These genes show considerable sequence similarity to the sel-12 gene of Caenorhabditis elegans, which has been postulated to function in the facilitated signalling by lin-12 and glp-1. In order to analyse the functional conservation of the presenilins, we introduced the human PS-1 cDNA, as well as clinical and deletion mutant proteins, into sel-12 mutant animals and tested their potential to rescue the egg-laying defect. Human PS-1 expressed from the sel-12 promoter fully rescued the sel-12 phenotype, whereas two missense mutations, C410Y and A246E, identified in pedigrees with FAD, exhibited a strongly decreased rescuing activity. The large hydrophilic loop and transmembrane domain 7 are required for the biological activity of PS-1. PS-1 protein was proteolytically cleaved in C. elegans as it is in human cells. A PS-1 splice variant (FAD mutation deltaexon9) that does not undergo proteolytic cleavage also substituted for sel-12. The conservation of function of human PS-1 and C. elegans sel-12 suggests that presenilin proteins are required, directly or indirectly, for the proper operation of the Notch signalling pathway. FAD-associated mutant proteins tested showed different rescuing activities, indicating that they might affect different functional or regulatory aspects of PS-1. Proteolytic processing is not a prerequisite for PS-1 function in C. elegans.     

Accumulation of human immunodeficiency virus-specific cytotoxic T lymphocytes away from the predominant site of virus replication during primary infection

Down-regulation of the initial burst of viremia during primary human immunodeficiency virus (HIV) infection is thought to be mediated predominantly by HIV-specific CD8+ cytotoxic T lymphocytes (CTL). This response is associated with major perturbations in the T cell receptor (TCR) repertoire. To investigate the failure of the cellular immune response to adequately control viral spread and replication and to prevent establishment of HIV infection, changes in the TCR repertoire and in the distribution of virus-specific CTL between blood and lymph node were analyzed in three patients with primary infection. By the combined use of clonotype-specific polymerase chain reaction and analysis of the frequency of in vivo activated HIV-specific CTL, it was shown that HIV-specific CTL clones preferentially accumulated in blood as opposed to lymph node. Accumulation of HIV-specific CTL in blood occurred prior to effective down-regulation of virus replication in both blood and lymph node. These findings should provide new insights into how HIV, and possibly other viruses, elude the immune response of the host during primary infection.     

Presence of pentoxifylline during T cell priming increases clonal frequencies in secondary proliferative responses and inhibits apoptosis

Naive T cells appear to be primed by specific Ag to differentiate into either effectors or memory cells. We have been analyzing the factors involved in this differential commitment in the priming of alloresponsive human T cells in vitro and have shown that the presence of a phosphodiesterase inhibitor, pentoxifylline (POX), during priming results in a decrease in the primary response and enhancement in the secondary proliferative response. We now show that the POX-mediated effect can be mimicked by dibutyryl cAMP. The secondary response enhancement is due to the effects of POX on the T cells rather than the APCs, because even fixed APCs can prime T cells in the presence of POX. POX affects T cells directly by increasing clonal frequency rather than the burst size of the secondary responders. The known inhibition of IL-2 production by POX is not responsible for this effect, because exogenous IL-2 supplementation does not block it. The presence of POX during priming alters the outcome of T cell activation, resulting in a lower frequency of cells expressing IL-2R alpha (CD25) and a decrease in their subsequent apoptosis, and this antiapoptotic effect is consistent with the enhanced commitment of T cells to secondary responsiveness by POX.     

Clofazimine and B669 inhibit the proliferative responses and Na+, K(+)-adenosine triphosphatase activity of human lymphocytes by a lysophospholipid-dependent mechanism

The relationship between the phospholipase-stimulating and immunosuppressive properties of the riminophenazine anti-mycobacterial agent clofazimine and its experimental analogue, B669, has been investigated in vitro. At concentrations of 0.6 microM and upwards, both riminophenazines, particularly B669, caused dose-related inhibition of mitogen- and alloantigen-stimulated uptake of tritiated thymidine by human mononuclear leucocytes (MNL), while in short-term assays both agents increased the release of lysophosphatidylcholine (LPC) and arachidonic acid from these cells. Arachidonate per se at a concentration of 20 microM did not affect mitogen-activated lymphocyte proliferation, while cyclooxygenase and 5'-lipoxygenase inhibitors, as well as water- and lipid-soluble oxidant-scavengers and anti-oxidant enzymes, failed to protect the cells against the anti-proliferative effects of clofazimine and B669. However, LPC caused dose-related inhibition of lymphocyte proliferation. Moreover, co-incubation of NML with alpha-tocopherol (vitamin E), a lysophospholipid complex-forming agent, or with lysophospholipase, protected the cells against clofazimine and B669, as well as against LPC. Na+, K(+)-adenosine triphosphatase was identified as the primary target of riminophenazine/LPC-mediated inhibition of lymphocyte proliferation. Excessive release of anti-proliferative lysophospholipids during clofazimine or B669 treatment of mitogen- or antigen-activated lymphocytes is the probable biochemical mechanism of the immunosuppressive activity of these agents.     

The immunogenic properties of human melanomas and melanoma-associated antigens recognized by cytotoxic T lymphocytes

During the last years significant progress has been achieved in the identification of melanoma-associated antigens (MAA) recognized by cytotoxic T lymphocytes (CTL). These antigens belong to three main groups: tumor-associated testis-specific antigens (MAGE, BAGE, GAGE and PRAME), melanocyte differentiation antigens (tyrosinase, Melan-A/MART-1, gp100, TRP-1 and TRP-2) and mutated or aberrantly expressed antigens (MUM-1, CDK4, beta-catenin, gp100-in4, p15 and N-acetylglucosaminyltransferase V). For the identification of these antigens, CTL cultures from mainly only 4 different melanoma patients have been used. These patients developed a strong anti-melanoma response resulting in long-lasting disease-free periods, pointing to the importance of the identification of highly immunogenic melanomas. In each of these patients, the immune response was observed against a unique set of 4 to 6 individual antigenic epitopes, on one hand suggesting the low immunogenicity of the individual antigens, and on the other pointing to the importance of the identification of additional highly immunogenic melanomas for the discovery of new MAA. The analysis of the available data on the immunogenic and protective properties of individual MAA confirms their low immunogenicity. In our study, we focused on the identification of especially highly immunogenic melanomas among a panel of 40 newly established melanoma cell lines. So far, only two such melanoma cell lines, FM3 and FM57 have been identified in this panel. The immunogenic properties of uncloned FM3 cells and several FM3 clones have been further investigated. It was found that the immunogenic properties of melanoma cells are mainly determined by the expression of progression-associated antigens as well as by ecto-ATPase, a molecule which is able to modulate cell adhesion. Cloning the cultures of PBL, stimulated with uncloned FM3 or with the highly immunogenic FM3 clone, FM3.29, has permitted us to identify the immune response against eight different MAA, five of these probably representing not previously described antigens. (Tab. 2, Fig. 2, Ref. 68.)     

Cell walls, peptidoglycans, and teichoic acids of gram-positive bacteria as polyclonal inducers and immunomodulators of proliferative and lymphokine responses of human B and T lymphocytes

In this study, the mitogenic and immunomodulating effects of bacterial cell wall preparations were investigated. Cell walls, peptidoglycans, and teichoic acids from Bacillus subtilis and Staphylococcus aureus Wood 46 activated both human T cells (supplemented with 10% monocytes) and B cells to proliferative and to produce leukocyte migration inhibitory factor. Similar results were obtained with adult and umbilical cord blood cells, suggesting that these bacterial preparations acted as mitogens. Cell walls and peptidoglycans had a modulating effect on purified protein derivative-induced and protein A-induced proliferation. In the presence of suboptimal concentrations of these stimulants, bacterial components enhanced the proliferative response. However, at optimal concentrations of purified protein derivative or protein A, bacterial components suppressed lymphocyte proliferation. Peptidoglycans solubilized by lysozyme activated B lymphocytes but not T cells. Solubilization had no effect on immunomodulating capacity.     

Proliferation and agglutinability of primary and transformed human epithelial cells in culture

Primary epithelial populations (HAM) were obtained by dissociation of the amniotic membrane stripped from human placentae. Agglutinability of cells from such normal populations and of cells from the transformed epithelial line WISH was then compared using concavanalin A as mediator. Extensive similar studies have previously been reported with cell strains isolated from other species. Freshly dissociated HAM cells from primary cultures agglutinated much less readily than did cells from WISH populations. Furthermore, the former exhibited a drastic decline in agglutinability as a function of time in suspension culture after trypsinization. Short-term exposure (60 h) of HAM cells in monolayer culture to 5-bromodeoxyuridine (BrdU) elicited heightened agglutinability detectable through 22 days in vitro. Addition of the protease inhibitors n-tosyl-L-lysyl-chloromethyl ketone (TLCK) or p-tosyl-L-arginine-methyl ester (TAME) to the culture medium inhibited proliferation of the WISH line by 40--50% while effecting only a 10-15% inhibition of HAM cells. These results also confirm data with other cell species indicating that high proteolytic activity at the surface of transformed cells may be related to the rapid proliferation rate.     

Immunologic injury in measles virus infection. III. Presence and characterization of human cytotoxic lymphocytes

Human peripheral blood leukocytes (PBL) from patients with chronic measles virus infection (SSPE) or from immune, adult humans (convalescent from acute childhood measles virus infection) are cytotoxic for target cells infected with measles virus as measured by a 51Cr assay. Specific release of 51Cr by immune PBL occurred both with and without human antibodies added to measles virus-infected cultures. Maximal killing in the absence of added antibodies to measles virus was usually detected only after 15 to 18 hr of incubation and with a high PBL to target ratio (100:1). When antibody to measles virus was added, PBL-mediated killing of virus-infected cells was not blocked. Instead, killing was enhanced and maximal lysis occurred with fewer PBL and a shorter incubation time. This cytotoxic reaction was inhibited in a dose-response manner upon the addition of Fab fragments of IgG containing antibodies to measles virus. On the average 4 to 5 x 105 antibody molecules bound per infected target cell before initiation of antibody-enhanced PBL killing. Depletion of either glass-adhering or E-rosette-forming cells did not reduce PBL killing of measles virus-infected target cells in either system. In contrast, removal of non-E rosette or of EAC rosette-forming population of PBL almost completely abrogated cytotoxicity. When Fc-bearing cells were removed, killing of virus-infected target cells was concomitantly reduced. Lysis of measles virus-infected target cells did not require histocompatibility between the PBL and the target cell. Further, immunospecific lymphocyte killing was not enhanced by such a histocompatibility fit. These experiments indicate first, that the effector PBL involved in lysis of measles virus-infected targets are not T cells but are probably K cells and, second, that PBL obtained from patients with SSPE are competent in killing measles virus-infected targets. Moreover, sera from SSPE patients did not contain a factor(s) that blocked PBL-mediated cytotoxicity.     

Direct activation of human CD8+ cytotoxic T lymphocytes by interleukin-18

Direct activation of human cytotoxic T lymphocytes (CTL) by interleukin (IL)-18 was observed in a system in which CTL effective against autologous tumor cells were generated. Peripheral blood mononuclear cells (PBMC) from tumor-bearing patients, after removal of natural killer (NK) cells, were cultured in a medium containing IL-1, -2, -4, and -6, with or without IL-18, and stimulated with autologous tumor cells. IL-18 increased the activity of the CTL and the proportion of autologous CD8+ T cells present after 28 days in the induction culture. When purified CD8+ T cells were cultured in the presence of IL-18 and IL-2 for 7 days, the CTL showed enhanced cytotoxic activity against autologous tumor cells. Moreover, a purified CD8+ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7-day incubation with IL-18. These results suggest that IL-18 may be useful to generate autologous CTL in humans and may thereby contribute to adoptive immunotherapy for tumors.     

MHC restriction of T-cell proliferative responses in Xenopus

The MHC restriction of Xenopus allogeneic MHC- and antigen-specific T-cell proliferative responses was assessed. Xenopus MHC-specific monoclonal antibodies that recognize class I and class II molecules were tested for inhibitory effects on the generation of secondary T-cell proliferative responses. Antigen-specific T-cell lines were inhibited by anti-class II but not anti-class I monoclonal antibodies. Secondary alloantigen-specific proliferative responses also demonstrated MHC class II restriction. Allogeneic MHC- and antigen-specific T-cell lines demonstrated differential sensitivity to anti-class II monoclonal antibodies directed at discrete class II epitopes. These results indicate that Xenopus T cells interact with antigen-presenting cells similarly to mammals, and directly confirm previous data indicating that MHC class II restriction of proliferative responses is present in amphibians.     

Extensive proliferative capacity of single isolated CD34 human cord blood cells in suspension culture

Nonadherent, low-density T-lymphocyte-depleted (NALT-) CD34 cells from normal human cord blood were assessed in suspension culture for the effects of recombinant cytokines on their proliferation, differentiation, and generation of myeloid progenitor cells. In this cell population, 82% of cells expressed c-kit protein as assessed by in situ hybridization, and their cloning efficiency was 85% when cells were plated at low cell numbers with combinations of growth factors. CD34 cells were sorted as 1, 5, or 10 cell(s) per well and also at 5000 cells per dish to initiate stromal-free suspension cultures in the presence of steel factor (SLF), interleukin (IL)-1 alpha, and IL-3. Forty-eight percent of the wells started with a single CD34 cell were positive for growth after 14 days, and the wells contained greater than 5 x 10(3) cells by 21-28 days. Progenitors were assayed weekly with cultures initiated with 1 or 5000 cells. While the fold expansion of nucleated cells was greater in cultures initiated with one cell per well (> 5000 compared to 791-fold expansion for 5000 cells), the fold expansion of progenitors was greater than 5000 cells were used to initiate cultures. Under optimal conditions, there was, respectively, a 160-, 164-, and 57-fold output of high proliferative potential colony-forming cells, granulocyte-macrophage colony-forming units, and erythroid burst-forming units/granulocyte erythroid macrophage megakaryocyte colony-forming units within 1-3 weeks for cultures initiated with 5000 CD34 cells compared with respective fold increases of 29, 16, and 1, for single-initiated cultures. These results demonstrate the expansion capacity of single CD34 cord blood cells and demonstrate that factors in addition to SLF, IL-1 alpha, and IL-3 are necessary for optimal expansion of progenitors from single isolated CD34 cells.