The pattern of gene expression in mouse Gr-1(+) myeloid progenitor cells.

To understand the pattern of gene expression in mouse myeloid progenitor cells, we carried out a genome-wide analysis of gene expression in mouse bone marrow Gr-1(+) cells using SAGE and GLGI techniques. We identified 22,033 unique SAGE tags with quantitative information from 73,869 collected SAGE tags. Among these unique tags, 64% match known sequences, including many genes important for myeloid differentiation, and 36% have no matches to known sequences and are likely to represent novel genes. We compared the expression of mouse Gr-1(+) and human CD15(+) myeloid progenitor cells and showed that the pattern of gene expression of these two cell populations had some similarities. We also compared the expression of mouse Gr-1(+) myeloid progenitor cells with that of mouse brain tissue and found a highly tissue-specific manner of gene expression in these two samples. Our data provide a basis for studying altered gene expression in myeloid disorders using mouse models.

Immunological enhancement of primary tumor development and its prevention.

While it has been known for decades that the growth of tumor transplants can be enhanced immunologically, the potential significance of these previous findings to the development of primary tumors and the mechanisms of tumor enhancement has remained obscure. This review will summarize recent experiments indicating that primary tumor development can be enhanced by active immunization. The evidence suggests that antibodies, B cells and CD4+ T cells can play a critical role in enhancing the development of primary, tumors, whereas endogenous interferon-gamma (IFNgamma) can counteract enhancement. Thus, we envision two possible functions of IFNgamma: (i) preventing B cell and antibody enhancement and (ii) counteracting tumor promotion independent of T and B cells.

Enhanced growth of primary tumors in cancer-prone mice after immunization against the mutant region of an inherited oncoprotein.

One major objective of tumor immunologists is to prevent cancer development in individuals at high risk. (TG.AC x C57BL/6)F1 mice serve as a model for testing the feasibility of this objective. The mice carry in the germline a mutant ras oncogene that has an arginine at codon 12 instead of glycine present in the wild-type, and after physical (wounding) or chemical promotion, these mice have a high probability for developing papillomas that progress to cancer. Furthermore, F1 mice immunized with Arg(12) mutant ras peptide in complete Freund's adjuvant (CFA) develop T cells within 10 d that proliferate in vitro on stimulation with the Arg(12) mutant ras peptide. Within 14 d, these mice have delayed-type hypersensitivity to the peptide. Immunization with CFA alone or with a different Arg(12) mutant ras peptide in CFA induced neither response. To determine the effect of immunization on development of tumors, mice immunized 3 wk earlier were painted on the back with phorbol 12-myristate 13-acetate every 3 d for 8 wk. The time of appearance and the number of papillomas were about the same in immunized and control mice, but the tumors grew faster and became much larger in the mice immunized with the Arg(12) mutant ras peptide. Thus, the immunization failed to protect against growth of papillomas. The peptide-induced CD4(+) T cells preferentially recognized the peptide but not the native mutant ras protein. On the other hand, mice immunized with Arg(12) mutant ras peptide and bearing papillomas had serum antibodies that did bind native mutant ras protein. Together, these studies indicate that active immunization of cancer-prone individuals may result in immune responses that fail to eradicate mutant oncogene-expressing tumor cells, but rather induce a remarkable enhancement of tumor growth.

B lymphocytes secreting IgG linked to latent transforming growth factor-beta prevent primary cytolytic T lymphocyte responses.

B lymphocytes secreting IgG linked to latent transforming growth factor (TGF)-beta (IgG-TGF-beta) prevent cytolytic T lymphocyte (CTL) responses to unrelated antigens in mixed lymphocyte cultures (MLC) so long as resting resident macrophages and functional Fc receptors are present. This was shown using IgG-secreting plaque-forming cells (PFC) to sheep erythrocytes (SRBC) obtained from popliteal lymph nodes of mice injected repeatedly in foot pads with SRBC. Remarkably, as few as approximately 300 PFC prevented CTL responses of 5 x 10(5) normal syngeneic spleen cells in MLC. Supranatants of short-term cultures of PFC also prevented CTL responses, and suppression was prevented by eliminating or dissociating IgG and TGF-beta present in supranatants or by antibody against active TGF-beta. Furthermore, the latency-associated peptide of latent TGF-beta was detected in approximately 10% of foci of IgG captured from single PFC, indicating that at least some B lymphocytes secrete IgG-TGF-beta as a complex. Resting resident macrophages (which do not produce latent TGF-beta) and functional Fc receptors were required for suppression, consistent with idea that IgG-TGF-beta is taken up through Fc receptors for IgG and that active TGF-beta, cleaved from latent TGF-beta of the complex, is delivered directly to potentially responding CTL. If CTL responses in man are similarly regulated by B lymphocytes, then an ongoing B cell response in patients with chronic viral infections or bearing immunogenic cancers may prevent effective therapeutic vaccination.

Synergy between T-cell immunity and inhibition of paracrine stimulation causes tumor rejection.

During tumor progression, variants may arise that grow more vigorously. The fate of such variants depends upon the balance between aggressiveness of the variant and the strength of the host immunity. Although enhancing host immunity to cancer is a logical objective, eliminating host factors necessary for aggressive growth of the variant should also be considered. The present study illustrates this concept in the model of a spontaneously occurring, progressively growing variant of an ultraviolet light-induced tumor. The variant produces chemotactic factors that attract host leukocytes and is stimulated in vitro by defined growth factors that can be produced or induced by leukocytes. This study also shows that CD8+ T-cell immunity reduces the rate of tumor growth; however, the variant continues to grow and kills the host. Treatment with a monoclonal anti-granulocyte antibody that counteracts the infiltration of the tumor cell inoculum by non-T-cell leukocytes did not interfere with the CD8+ T-cell-mediated immune response but resulted in rejection of the tumor challenge, indicating a synergy between CD8+ T-cell-mediated immunity and the inhibition of paracrine stimulation.

Autoantibodies produced spontaneously by young 1pr mice carry transforming growth factor beta and suppress cytotoxic T lymphocyte responses.

Young MRL/MPJ-lpr (lpr) mice 8-12 wk old challenged with alloantigen had significantly lower specific cytolytic T lymphocyte (CTL) responses than control MRL/MPJ +/+ mice. Serum from lpr mice compared with serum from ++ or normal C3H mice powerfully suppressed CTL responses in mixed lymphocyte cultures (MLC); absorbing lpr serum on protein G, adding antibody against transforming growth factor beta (TGF-beta) to cultures or dissociating immunoglobulin G (IgG) and TGF-beta before additions to cultures prevented suppression. Apparently autoantibody, similar to IgG produced by normal mice in response to immunization, carries TGF-beta which suppresses CTL responses in vivo and in vitro.

Tumors with reduced expression of a cytotoxic T lymphocyte recognized antigen lack immunogenicity but retain sensitivity to lysis by cytotoxic T lymphocytes.

A murine solid tumor was transfected to express various levels of an allogeneic major histocompatibility complex class I gene (K216), in order to test the effect of the level of antigen expression on immunogenicity and sensitivity to lysis by cytotoxic T lymphocytes (CTL). The growth rates of clones of tumor cells expressing different levels of the transfected gene were similar in vitro and in nude mice. Although all tumor cells, including cells freshly isolated from growing tumors, were equally sensitive to lysis by specific CTL, only tumor cells expressing the highest level of the K216 antigen stimulated CTL and were rejected by normal mice. In contrast, tumor cells expressing lower levels of antigen failed to immunize for CTL and grew progressively in normal mice, despite retaining expression of the transfected gene and remaining fully sensitive to CTL-mediated lysis; thus, the threshold of antigen needed to stimulate CTL responses was considerably higher than that needed to lyse tumor cells. Reduction of K216 antigen expression from 100-fold to 40-fold above background, impaired significantly the ability of the tumor cells to induce a K216-specific immune response, while tumor cells expressing K216 at levels 2-fold above background were as susceptible to CTL-mediated lysis as tumor cells expressing 50-fold more antigen. The important implication of these findings is that some tumors occurring in nature may not be immunogenic but nevertheless express antigens which are potential targets for immune therapy.

A first or dominant immunization. I. Suppression of simultaneous cytolytic T cell responses to unrelated alloantigens.

A first or dominant immunization with one antigen markedly inhibited specific cytolytic T lymphocyte (CTL) responses to a second unrelated alloantigen without suppressing antibody responses to other antigens. Suppression was induced rapidly, became systemic, and could be transferred passively with only serum. Suppression did not result from elimination of cells capable of responding to the second antigen. The mechanisms responsible for this "priority of the first response" may be the same that help protect the fetus during pregnancy, promote renal allograft survival after multiple blood transfusions, and prevent effective CTL-mediated immunity to variants of tumor cells or infectious agents that arise during tumor progression or chronic infections.

A first or dominant immunization. II. Induced immunoglobulin carries transforming growth factor beta and suppresses cytolytic T cell responses to unrelated alloantigens.

Fresh sera from mice immunized by bearing an immunogenic tumor or by repeated injections of allogeneic spleen cells or xenogeneic erythrocytes powerfully suppress cytolytic T cell responses in one-way mixed lymphocyte cultures. Suppression is not antigen specific, though is mediated by immunoglobulin (Ig)G specific for the immunizing antigen. Suppression caused by IgG mimics that caused by active transforming growth factor beta (TGF-beta). IgG associates with or carries latent TGF-beta; however, suppression caused by the complex of IgG-TGF-beta requires macrophages (M phi), whereas active TGF-beta alone does not. Also, IgG dissociated from TGF-beta does not cause suppression, suggesting that M phi may take up Ig-TGF-beta, process the complex, and deliver active TGF-beta to lymphocytes. Indeed, suppression by immune serum was prevented by antibody to Fc receptors, by saturating Fc receptors with heterologous IgGs, and by antibodies against TGF-beta. The overall findings reveal a previously unrecognized regulatory circuit whereby IgG produced in response to one antigen nonspecifically downregulates cytolytic T lymphocyte responses to unrelated antigens. The findings introduce the intriguing possibility that TGF-beta delivered by IgG and processed by M phi may mediate important biological effects in processes such as wound healing, tumor growth, and some autoimmune diseases.

Flow cytometric analysis of lymphocyte surface markers following a 1-Gy dose of gamma radiation.

Flow cytometric analysis of lymphocytes labeled with monoclonal antibodies was undertaken in order to compare the mean fluorescent intensity of several surface membrane antigens (markers) from irradiated and nonirradiated blood samples. Whole blood subjected to a 1-Gy (100-rad) dose of 1.25 MeV gamma radiation from a 60Co radioisotope source was compared with nonirradiated blood drawn simultaneously from the same healthy subject. Twenty runs were performed in which the following T and B lymphocyte and natural killer surface markers were analyzed: CD2, CD3, 13, CD4, CD29, CD45RA, CD8, CD56, and CD19. The data demonstrate a radiation-induced decrement in the mean fluorescent intensity of the high molecular weight markers CD45RA and CD56 (decrement is -3.3% and -7.2%, respectively). The statistical validity of these values was confirmed using paired t-tests, which yielded p values of p < 0.02 (CD45RA) and p < 0.01 (CD56). The fluorescent intensity is proportional to the number of intact binding sites on the lymphocyte surface and the observed decrement directly infers that damage to some sites occurred. These results illustrate a measurable effect on the lymphocyte membrane at a radiation dose at which many lymphocytes will survive, yet may be immunologically altered. This study may have important implications for personnel exposed to ionizing radiation, such as astronauts on long duration missions and radiation workers involved in accidental exposures.

CD4-positive and B lymphocytes in transplantation immunity. I. Promotion of tumor allograft rejection through elimination of CD4-positive lymphocytes.

The elimination of CD4+ cells by anti-CD4 antibody caused regression of a malignant solid tumor allograft that does not lose a cytotoxic T lymphocyte-defined target antigen during tumor progression and requires specific CD8+ CTL for tumor rejection. Treatment with anti-CD4 antibody was effective when started 1-2 weeks after tumor challenge and was at least as effective as treating with anti-CD3 antibody or specific immunization with the antigen expressed on malignant or nonmalignant cells. None of these treatments caused rejection of tumors that were larger than 1 cm3 or had been growing for 3 weeks or longer in the host. Mice bearing large and long-established tumors treated with anti-CD4 antibody rejected a new tumor challenge but failed to reject the long-established tumor. Similarly, mice with established tumors mounted effective CTL responses to reject skin grafts but failed to reject tumors which expressed the same antigen. Treatment with anti-CD4 antibody eliminated primary T lymphocyte dependent antibody responses but failed to suppress ongoing antibody responses to continuous antigenic stimulation. Possibly, the effectiveness of early treatment and the failure of later treatment with anti-CD4 antibody results indirectly from the effect treatment has on B lymphocytes.

CD4+ and B lymphocytes in transplantation immunity. II. Augmented rejection of tumor allografts by mice lacking B cells.

To test the importance of B lymphocytes in immunity to major histocompatibility complex class I alloantigens, B cell-deficient mice were generated by reconstituting severe combined immunodeficiency mice, which lack functional B and T lymphocytes, with T cells or with both T and B cells. The reconstituted mice were challenged with a cancer that expresses an MHC class I alloantigen at a low level and is susceptible to killing by CD8+ cytotoxic T lymphocytes. Tumors grew more slowly in and were rejected more frequently by the mice lacking B cells. Understanding the mechanism by which B cells suppress tumor allograft rejection may lead to new approaches for suppressing immune attack on transplanted tissues.

Development of a fiber-optic sensor for trace metal detection in aqueous environments.

The availability and speciation of a number of metals widely dispersed in the aquatic environment intimately affect the biogeochemistry of the ocean and its inhabitants. Much research has been focused on the development of analytical methodologies to elucidate better the background concentrations, variability, and contaminant effects of metal species. The purpose of this research is to investigate the viability of a fiber-optic sensor that will be a sensitive and selective probe for trace metals in natural waters.

Quantitative measures of sympathetic skin response in diabetes: relation to sudomotor and neurological function.

The sympathetic skin response (SSR) at the foot to a deep inspiration was measured in 68 randomly selected diabetic patients and 46 age matched normal subjects and compared with other quantitative measures of neurological and sudomotor function. SSR was obtained in all but three diabetic patients. The upper limit of normal for the onset latency was 2202 ms and the lower limit for the amplitude of the first wave 92 microV. Ten diabetic patients had measurable but prolonged latencies, and 11 had measurable but low amplitudes. There were no significant associations between latency, height, and age, but in insulin dependent patients there was a significant diminution of response amplitude with increasing duration of diabetes. Latency was weakly associated with Marstock thermal thresholds, respiratory RR variation, and common peroneal nerve conduction velocity. SSR amplitude was associated with the density of pilocarpine activatable sweatspots in the same region of the foot. Patients with abnormal latencies were significantly older and had reduced thermal sensation than those with normal latencies. Median coefficients of variation for repeat testing in diabetic patients were 9% for latency and 13% for amplitude. The test is objective and reproducible, but latency measurements reflect conduction in a long multineuronal pathway and are not purely a measure of peripheral C fibre function; amplitude measurements reflect the density of spontaneously activable sweat glands and are therefore a valid measure of peripheral sympathetic activity, though they depend more on temperature than do latencies (mean change over the range 32-34 degrees C; 8.5% degrees C for amplitude, -2.5%/degrees C for latency).

Portable infrared pupillometry using Pupilscan: relation to somatic and autonomic nerve function in diabetes mellitus.

The relationship between dynamic pupillary function and peripheral nerve function was studied in 85 randomly-selected diabetic patients and 67 age-matched normals using a portable infrared pupillometer (Pupilscan Version 5). Seven measurements were chosen to represent different components of the pupillary constriction-redilatation curve after a standardized light stimulus. Constriction latency was significantly prolonged in diabetic patients (p = 0.05), as was time to 63% redilatation (p = 0.001). Thermal thresholds at the feet weakly correlated with relative reflex amplitude (warm: r = -0.22, p = 0.05; cool: r = -0.23, p = 0.05), but vibration perception thresholds were more strongly associated with constriction and redilatation velocity (r = -0.42, p = 0.001; r = -0.28, p = 0.03). Among the cardiovascular autonomic function tests, only respiratory R-R variation correlated with constriction velocity (r = 0.47, p < 0.001), and Valsalva ratio with redilatation velocity (r = 0.25, p = 0.04), but postural systolic blood pressure change was also correlated with reflex amplitude and latency time (r = -0.42, p < 0.001; r = 0.41, p = 0.001). There were no significant associations with three measures of sweating function in the feet. Pupil measurements were abnormal in 4-11% of diabetic patients, while other neurological tests were abnormal in 8-35%, consistent with the length-dependence of diabetic neuropathy. Median coefficients of variation were 2.0-7.2% in diabetic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells.

Inoculated immunogenic cancer cells after initial growth are potentially rejected by specific host immunity; however, the outcome of the interaction between host and inoculated cancer cells is a function of multiple factors including the route of inoculation, the number of cells, the density of antigens on the injected cancer cells, and the state of the immune system of the host. In the present study, we have examined a different kind of variable: the stroma that inoculated tumor cells initially reside in. The impetus to examine this factor arises from observations that cancer cells from several lines inoculated as fragments of solid tumors often grow progressively, whereas the same number or more than 10-fold larger numbers of identical type cells injected as a suspension are rejected, even though fragments or suspended cells are both tumorigenic at the same doses in nude mice. In the present studies, we found that: (a) indeed, cancer cells inoculated as fragments were more tumorigenic than cancer cells in suspension; (b) the tumorigenicity of suspended cancer cells was increased by injection of the cells into polyurethane sponge implants; (c) cancer cells were more tumorigenic embedded in syngeneic stroma than in transgenic antigenic stroma expressing the K216 major histocompatibility complex class I antigen; and (d) antigenic, bone marrow-derived, stromal components (presumably passenger leukocytes) were sufficient to cause rejection of immunogenic but antigenically unrelated cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in cholinergic sweat gland activation in diabetic neuropathy identified by computerised sweatspot analysis.

Peripheral small-fibre denervation has been reported to result in decreased activation of eccrine sweat glands to muscarinic cholinergic agents. Using computerised image-analysis of pilocarpine-activated sweatspot prints of a 4 cm2 area of the dorsum of the foot in 79 randomly selected diabetic patients we have identified a group of neuropathic patients (18%) with decreased sweatspot activation (less than 20/cm2), and a smaller group (6%) of younger patients with less marked neuropathy who had increased activation (greater than 132/cm2), probably resulting from denervation supersensitivity. The associations between sweatspot density and other conventional tests of peripheral nerve function were weak. The prevalence of abnormal sweatspot density, 24%, was similar to that of other tests, except thermal thresholds at the feet (35-37%), which were not correlated with sweatspot activation, suggesting that diabetic neuropathy has differing effects on afferent and efferent small fibres. The method is rapid and reproducible (median coefficient of variation 14%) and its ability to identify patients with increased, as well as decreased, peripheral nerve function may be of value in the characterisation and longitudinal follow-up of small-fibre abnormalities in diabetes.

Cytokines and cancer: experimental systems.

The transfer of certain cytokine genes into cancer cells can provide very powerful suppression of tumor growth in the absence of any toxic side effects. Some of these cytokines, such as interleukin-4, granulocyte colony-stimulating factor and tumor necrosis factor, can mediate powerful immune suppression even in T-cell-deficient animals and appear to be effective for poorly or non-antigenic tumors. However, approaches must be found to induce or deliver cytokines locally at the tumor site.

Assessment of basal and stimulated sweating in diabetes using a direct-reading computerized sudorometer.

Abnormalities of eccrine sweating are thought to be common in diabetes. We describe a ventilated-capsule sudorometer for the continuous measurement of basal and stimulated sweat secretion. It is sensitive (detecting as little as 200 ng water vapour), precise, and stable. Since it measures dewpoint rather than relative humidity, it can be calibrated to read sweat volumes directly and independently of ambient temperature and humidity. Preliminary studies using this technique show that basal skin water loss is significantly diminished in patients with established diabetic neuropathy (0.91 +/- 0.18 g (+/- SD) cm-2 h-1) compared with normal subjects (1.21 +/- 0.39 g cm-2 h-1; p = 0.04) and non-neuropathic diabetic subjects (1.32 +/- 0.48 g cm-2 h-1; p = 0.04), and that local sweating induced by iontophoresis of 10 g l-1 acetylcholine is significantly reduced in diabetic subjects up to 5 min of recording (0.95 +/- 0.43 vs 1.26 +/- 0.40 mg; p = 0.02). In neuropathic subjects both low- and high-amplitude responses are seen, the latter probably representing denervation supersensitivity. Further studies with sensitive sudorometry should enable the mechanisms of these abnormal responses to be established.

Major histocompatibility complex class I and unique antigen expression by murine tumors that escaped from CD8+ T-cell-dependent surveillance.

The rejection of murine UV-induced skin cancers by normal mice is a striking example of powerful immune surveillance of the normal host against malignant cells. In this study, we show that UV-induced regressor tumors regularly grew progressively and killed mice that were depleted of CD8+ T-cells. Depletion of CD4+ T-cells had no effect, suggesting that CD8+ but not CD4+ T-cells were required for this immune surveillance. To determine whether change in major histocompatibility complex (MHC) class I expression was a frequent event that caused low immunogenicity of tumors or facilitated escape from immune destruction, recently isolated murine tumors of varying degrees of immunogenicity, including highly immunogenic UV-induced regressor, less immunogenic UV-induced progressor, and poorly immunogenic spontaneous progressor tumors, were compared. There was no correlation between the ability of a tumor to grow progressively in a normal immunocompetent host and the level of constitutive class I expression or the level of expression induced in vitro by gamma interferon. (Only 1 of more than 20 progressor tumors analyzed showed complete loss of a MHC class I molecule.) Some progressor variants showed loss of a unique tumor-specific cytotoxic T-lymphocyte-defined antigen, consistent with earlier evidence of antigen loss providing a mechanism for tumor escape. However, most of the host-selected progressor variants retained both MHC class I antigens and the unique tumor antigens that we could detect with cytotoxic T-lymphocyte clones, suggesting that mechanisms other than loss of MHC class I or of the unique target antigen may be involved in escape of some tumors from a highly effective CD8-dependent host surveillance.