Margaret Prechel

Mechanisms of immune suppression in patients with head and neck cancer : Influence on the immune infiltrate of the cancer

Freshly excised human head and neck cancers (219 primary cancers; 64 metastatic lymph node cancers) were analyzed for the immune inhibitory mediators released from the cancer tissues and the immune infiltrate within the tumor. Significant levels of the immune inhibitory mediators transforming growth factor‐β (TGF‐β), prostaglandin E2 (PGE2) and interleukin‐10 (IL‐10) were released from these cancers. Also released was granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), whose secretion was associated with an intratumoral presence of CD34+ cells. We have previously shown that CD34+ cells within human head and neck cancers are immune inhibitory granulocyte‐macrophage progenitor cells. The presence of TGF‐β, PGE2 and IL‐10 was associated with a reduced content of CD8+ T‐cells within the cancers. The CD4+ cell content appeared to be less affected by these immune inhibitory mediators. Instead, parameters indicative of CD4+ cell function (p55 IL‐2 receptor expression, release of IL‐2 and IFN‐γ) were diminished in cancers that released higher levels of TGF‐β, IL‐10 and GM‐CSF and had a higher CD34+ cell content. Furthermore, metastatic cancers released higher levels of the soluble immune inhibitory mediators and lower levels of IFN‐γ and IL‐2 than did primary cancers, although CD34+ cells were similarly present in both primary and metastatic cancers. Our results show that human head and neck cancers have a multiplicity of non‐mutually exclusive mechanisms of immune suppression that are most prominently associated with reduced CD8+ cell influx and reduced influx and altered function of intratumoral CD4+ cells.

Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocyte-macrophage colony-stimulating factor and contained CD34+ natural suppressor cells

Human head and neck squamous cell carcinomas (HNSCC) that produce high levels of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) have been shown to contain CD34+ natural suppressor cells that inhibit the activity of intratumoral T‐cells. The present study evaluated whether GM‐CSF production and the presence of CD34+ cells within primary HNSCC would translate into increased recurrence, metastasis or cancer‐related death during the 2 years following surgical excision. Freshly excised primary HNSCC of 20 patients that subsequently developed disease, and of 17 patients that remained with no evidence of disease were analyzed for production of GM‐CSF and for CD34+ cell content. The cancers of patients that subsequently developed recurrences or metastatic disease produced almost 4‐fold the levels of GM‐CSF and had approximately 2.5‐fold the number of CD34+ cells as did cancers of patients that remained disease‐free. In a second method of analysis, the prognostic significance of high vs. low GM‐CSF and CD34+ cell values was evaluated. These analyses showed that patients whose cancers produced high GM‐CSF levels or had a high CD34+ cell content had a disproportionately high incidence of recurrence or metastatic disease (94% and 100%, respectively), while the majority of patients whose primary cancers produced low levels of GM‐CSF or had a low CD34+ cell content remained disease‐free (16% and 19%, respectively). Our results indicate that the presence of CD34+ cells in GM‐CSF‐producing HNSCC is associated with a poorer prognosis for the cancer patients and suggest the utility of these parameters as prognostic indicators of outcome. Mechanistically, our results suggest that the presence of immune suppressive CD34+ cells in GM‐CSF‐producing HNSCC leads to increased tumor recurrence or metastasis.Int. J. Cancer 74:69–74.

Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands.

While heparin has been used almost exclusively as a blood anticoagulant, important literature demonstrates that it also has broad anti-inflammatory activity. Herein, using low anti-coagulant 2-O,3-O-desulfated heparin (ODSH), we demonstrate that most of the anti-inflammatory pharmacology of heparin is unrelated to anticoagulant activity. ODSH has low affinity for anti-thrombin III, low anti-Xa, and anti-IIa anticoagulant activities and does not activate Hageman factor (factor XII). Unlike heparin, ODSH does not interact with heparin-platelet factor-4 antibodies present in patients with heparin-induced thrombocytopenia and even suppresses platelet activation in the presence of activating concentrations of heparin. Like heparin, ODSH inhibits complement activation, binding to the leukocyte adhesion molecule P-selectin, and the leukocyte cationic granular proteins azurocidin, human leukocyte elastase, and cathepsin G. In addition, ODSH and heparin disrupt Mac-1 (CD11b/CD18)-mediated leukocyte adhesion to the receptor for advanced glycation end products (RAGE) and inhibit ligation of RAGE by its many proinflammatory ligands, including the advanced glycation end-product carboxymethyl lysine-bovine serum albumin, the nuclear protein high mobility group box protein-1 (HMGB-1), and S100 calgranulins. In mice, ODSH is more effective than heparin in reducing selectin-mediated lung metastasis from melanoma and inhibits RAGE-mediated airway inflammation from intratracheal HMGB-1. In humans, 50% inhibitory concentrations of ODSH for these anti-inflammatory activities can be achieved in the blood without anticoagulation. These results demonstrate that the anticoagulant activity of heparin is distinct from its anti-inflammatory actions and indicate that 2-O and 3-O sulfate groups can be removed to reduce anticoagulant activity of heparin without impairing its anti-inflammatory pharmacology.

Treating tumor-bearing mice with vitamin D3 diminishes tumor-induced myelopoiesis and associated immunosuppression, and reduces tumor metastasis and recurrence

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colonystimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1α,25-dihydroxyvitamin D3 reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D3 reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D3 treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D3 after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D3 treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.

Rivaroxaban - an oral, direct Factor Xa inhibitor - has potential for the management of patients with heparin-induced thrombocytopenia

Rivaroxaban is an oral, direct activated Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. Currently available anticoagulants include unfractionated heparin (UFH) and low molecular weight heparins (LMWHs); however, their use can be restricted by heparin‐induced thrombocytopenia (HIT). HIT is usually caused by the production of antibodies to a complex of heparin and platelet factor‐4 (PF4). This study was performed to evaluate, in vitro, the potential of rivaroxaban as an anticoagulant for the management of patients with HIT. UFH, the LMWH enoxaparin, fondaparinux and the direct thrombin inhibitor argatroban were tested to enable comparative analyses. Rivaroxaban did not cause platelet activation or aggregation in the presence of HIT antibodies, unlike UFH and enoxaparin, suggesting that rivaroxaban does not cross‐react with HIT antibodies. Furthermore, rivaroxaban did not cause the release of PF4 from platelets and did not interact with PF4, unlike UFH and enoxaparin. These findings suggest that rivaroxaban may be a suitable anticoagulant for the management of patients with HIT.

Activation of platelets by heparin-induced thrombocytopenia antibodies in the serotonin release assay is not dependent on the presence of heparin.

Summary.  The serotonin release assay (SRA) tests for antibodies responsible for heparin‐induced thrombocytopenia (HIT). By definition, SRA‐positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA‐positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T‐gel chromatography demonstrated differences among fractions of enzyme‐linked‐immunosorbent assay (ELISA)‐positive HIT antibodies within individual specimens. Certain ELISA‐positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.

Correlations of serotonin and its metabolites in individual rat pineal glands over light: Dark cycles and after acute light exposure

Profiles of pineal indolealkylamines were estimated by high performance liquid chromatography and were correlated in individual glands of male rats sacrificed over several light:dark cycles and after acute exposure to light at night. A significant and positive correlation of 5HIAA vs 5HT in individual glands over both normal and experimental lighting conditions suggested that oxidative deamination is not a major factor in photic regulation of pineal 5HT levels and that the formation of 5HIAA is dependent on substrate availability. Regression analysis of other indole constituents revealed that there was a positive and significant correlation between 5HT vs N-acetylserotonin, but not between 5HT vs melatonin and N-acetylserotonin vs melatonin in individual glands during the dark phase of a light:dark cycle. We propose that this effect may be related to a pulsatile release of melatonin into the blood stream and is the result of sampling glands at different stages in the storage/release of melatonin.

Simple scoring system for early management of heparin-induced thrombocytopenia.

This study was performed to develop a simple scoring system to aid in the early clinical management of patients suspected of heparin-induced thrombocytopenia (HIT) with regard to decisions for continued heparin therapy. The system was designed to arrive at low (0) or possible (1) probability scores without knowledge of laboratory test results (except platelet counts) to avoid delays. As the safest clinical approach is to discontinue heparin, intermediate and high scores were combined. Critically ill VA hospital patients (n = 100) with a ≥30% fall in platelet count were assessed by platelet aggregation (PA), 14C-serotonin release assay (14C-SRA), and GTI ELISA. In this population, 53% were scored 1 and of these 43% were positive by laboratory test. Emphasizing the decision to discontinue heparin, the clinical signs of HIT were paramount for the immediate determination of a diagnosis of HIT without dependence on a positive laboratory test.

The Laboratory Diagnosis and Clinical Management of Patients with Heparin-Induced Thrombocytopenia : An Update

Heparin-induced thrombocytopenia (HIT) is a serious adverse effect of heparin exposure that can progress to severe thrombosis, amputation, or death. HIT is an immune response in which antibodies cause platelet activation, platelet aggregation, the generation of procoagulant platelet microparticles, and activation of leukocytes and endothelial cells. Early diagnosis based on a comprehensive interpretation of clinical and laboratory information is important to improve clinical outcomes. However, limitations of the laboratory assays and atypical clinical presentations can make the diagnosis difficult. Clinical management of patients with HIT is with a non-heparin anticoagulant such as a direct thrombin inhibitor or danaparoid followed by a vitamin K antagonist for long-term treatment. The new anti-factor Xa drugs (fondaparinux, rivaroxaban, apixaban) and other non-heparin antithrombotic agents can potentially be used for the treatment of HIT if clinically validated. Important drug-specific limitations and dosing and monitoring guidelines must be respected for patient safety. Issues still exist regarding the optimal clinical management of HIT.

Vitamin D3 treatment of tumor bearers can stimulate immune competence and reduce tumor growth when treatment coincides with a heightened presence of natural suppressor cells

By secreting granulocyte-macrophage colony-stimulating factor (GM-CSF), Lewis lung carcinoma tumors induce immune suppressive granulocyte-macrophage progenitor cells. Treating mice having established tumors and high levels of suppressor activity with vitamin D3 eliminated suppressor activity, increased anti-tumor immunity, induced an immune stimulatory cell population, and reduced tumor growth. When instead, the vitamin D3 treatment was initiated earlier, when implanted tumors first became detectable and when natural suppressor activity was less prominent, the treatment had no effect. Thus, vitamin D3 treatment can stimulate the immune competence of tumor bearers when treatment is targeted to coincide with a heightened presence of GM-CSF-induced suppressor cells.