Elasticity transition and loop formation in vibrated bead chains: a simulation of polymer chains.

By measuring the distribution function of the end-to-end distance, we find that strongly shaken bead chains exhibit many properties, such as the rigid-rod-to-Gaussian chain transition, scaling, fast drop of loop formation probability in the short-chain regime, and enhancement of loop formation probability for kinked chains, of long-chain polymers. Though there is difference in local details between our chains and the worm-like chains, our results are consistent with recent calculations based on the worm-like chain model in many respects.

Relationship of diabetic macular oedema with glycosylated haemoglobin.

PURPOSE: To evaluate the correlation between glycosylated haemoglobin (HbA1c) and central foveal thickness as measured by optical coherence tomography (OCT) in patients with diabetes. METHODS: Retrospectively review of medical records of central foveal thickness as measured by OCT and laboratory data of glycosylated haemoglobin. HbA1c was compared with foveal thickness measured by OCT within the preceding 3 months. Clinically significant macular oedema (CSME) was diagnosed if central foveal retinal thickness was greater than 325 mum in OCT. RESULTS: One hundred and two eyes of 102 patients were included in this cross-sectional study. Univariate analysis revealed that the CSME diagnosed by OCT in diabetes was not statistically significant with sex, right or left eye, DM duration over 10 years or not, and AC sugar level (over 140 or not). The HbA1C level (8 or over) and age (50 or less) showed a significant (P=0.005 and 0.006, respectively) and positive association with macular thickness in OCT. A trend towards higher risk was seen for factors of age or=8%. CONCLUSIONS: Patients with HbA1c of 8 or above had an increase in macular thickness in type 2 diabetic eyes and there was a statistical significant correlation between younger age, shorter DM duration and thicker macular thickness. Strict sugar control decreased the risk of diabetic macular retinopathy, and OCT could be an excellent detector of early diabetic macular oedema.

Phase I trial of sequential cyclophosphamide, cyclosporin A, and interferon-alpha in patients with cancer: attempt to induce autologous graft-versus-host reaction to elicit an antitumor response.

Previous reports of autologous bone marrow transplant (auto-BMT) have demonstrated that myeloablative therapy followed by cyclosporin A (CsA), with and without interferon (IFN), can generate autoreactive cytotoxic T lymphocytes (auto-CTL) with potential therapeutic benefit. This is the first report of an attempt to generate auto-CTL using CsA and IFN after a non-myeloablative regimen. Cyclophosphamide (CTX) 1,200 mg/m2 i.v. day 1 was followed by CsA and IFN-alpha days 2-28, administered in a sequential three-step Phase I dose-escalation scheme. Patients were evaluated twice weekly for clinical evidence of graft-versus-host (GVH) reaction. Peripheral blood mononuclear cells (PBMCs) were obtained before treatment, at time of clinical GVH reaction, and days 21 and 28, and analyzed for auto-CTL, natural killer (NK) cell, and lymphokine-activated killer (LAK) cell activity. Patients also underwent punch skin biopsy at the time of clinical GVH reaction or day 21 to identify histologic evidence of GVH. Fourteen patients completed therapy and were evaluable for immunologic studies and anti-tumor response. No increase in auto-CTL, NK cell, or LAK cell activity was seen. Clinical or histologic evidence of GVH reaction did not occur. We conclude that this myelosuppressive dose of CTX combined with CsA and IFN is unable to generate clinical or immunologic evidence of an auto-GVH reaction. Further efforts are warranted to evaluate other therapeutic attempts to generate auto-CTL with anti-tumor activity based on preliminary results of clinical benefit in auto-BMT.

Spontaneous remission of renal cell carcinoma: a case report and immunological correlates.

Spontaneous remission of renal cell carcinoma is a rare occurrence for which immunological factors have been implicated as a possible mechanism. We report a case of spontaneous remission of metastatic renal cell carcinoma in which we assayed various immune parameters. At remission we found no enhancement of natural killer cell, lymphokine-activated killer cell or lymphocyte proliferative response.

Induction of murine peritoneal macrophage colony-forming cells by peritoneal administration of macrophage inflammatory protein-1 alpha.

Peritoneal injection of thioglycollate medium (TM) to mice results in a dramatic increase in total number of peritoneal macrophages within 48 to 72 hours. Unlike resident macrophages, a fraction (10 to 20%) of these newly arrived young macrophages, designated as macrophage colony-forming cells (M-CFC), are highly proliferative and formed macrophage colonies in vitro in the presence of either macrophage or granulocyte-macrophage colony-stimulating factor (M-CSF or GM-CSF). Using a reverse transcriptase polymerase chain reaction (RT-PCR) technique, peritoneal exudate macrophages (PEM) obtained 2 to 5 days after a single TM injection actively expressed mRNA for recombinant murine macrophage inflammatory protein-1 alpha (rmMIP-1 alpha). Yet none or only a trace amount of mRNA for MIP-1 alpha was detected in normal resident macrophages or PEM obtained 7 days after TM treatment. The effect of rmMIP-1 alpha on the induction of exudate M-CFC was investigated. Multiple intraperitoneal (IP) administration of rmMIP-1 alpha caused a marked increase in the total number of peritoneal M-CFC and macrophages similar to but weaker than the increase in TM-injected mice. The total number of neutrophils, mast cells, and eosinophils also increased, but with different kinetics, following multiple injections of rmMIP-1 alpha. rmMIP-1 alpha alone did not stimulate the proliferation of M-CFC, nor did it potentiate their responsiveness to either rmGM-CSF or recombinant human (rh) M-CSF in vitro. Taken together, our results suggest that MIP-1 alpha released by exudate macrophages is a major chemoattractant responsible for the migration of M-CFC from the circulation to the peritoneal cavity during a TM-induced inflammatory response.

Preclinical studies for adoptive immunotherapy in bone marrow transplantation. Generation of anti-CD3 activated cytotoxic T cells from normal donors and autologous bone marrow transplant candidates.

In order to obtain T cells for adoptive immunotherapy after autologous bone marrow transplantation (ABMT) for patients with resistant hematological malignancies, a culture system was developed for growing T cells and inducing non-MHC-restricted cytotoxicity using anti-CD3 monoclonal antibody (OKT3) activation. In this investigation, we show that (1) peripheral blood lymphocytes or bone marrow mononuclear cells (BMMNC) from normal donors and cancer patients can be activated with OKT3 and grown in interleukin-2; (2) normal BMMNC activated with OKT3/IL-2 exhibited non-MHC-restricted cytotoxicity and surface markers comparable to that exhibited by normal PBL activated with OKT3/IL-2; (3) both proliferation and cytotoxic functions were IL-2-dependent; (4) PBL activated with OKT3/IL-2 after cryogenic storage grew and killed comparable to PBL activated with OKT3/IL-2 prior to cryopreservation; (5) OKT3/IL-2-activated PBL and BMMNC obtained from 5 patients with non-Hodgkin's lymphomas (NHL) and 1 patient with acute myelogenous leukemia (AML) increased cell numbers 41-75-fold in 2 weeks of culture; 5 of 6 patients with NHL or AML had PBL and BMMNC that exhibited cytotoxic activity; and (6) contaminating leukemic cells did not overgrow in OKT3/IL-2-activated cultures and could no longer be detected on cytospin specimens after 3 weeks of culture. These data show that T cells in PBL or BMMNC from ABMT candidates can be activated with OKT3/IL-2 for adoptive immunotherapy in combination with ABMT.

Downregulation of M-CSF receptors by lipopolysaccharide in murine peritoneal exudate macrophages is mediated through a phospholipase C dependent pathway.

Murine peritoneal exudate macrophages (PEM) co-express granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF) receptors, among others. Treatment of PEM with lipopolysaccharide (LPS) or tumor-promoting phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]) induces a rapid but transient loss of M-CSF receptors in PEM. GM-CSF receptors are not affected by this treatment. The loss of M-CSF receptors induced by LPS can be inhibited by neomycin and compound 48/80, two potent phospholipase C (PLC) inhibitors, but not by phospholipase A2, calpain, protein kinase C (PKC) or protease inhibitors. On the other hand, the loss of M-CSF receptors induced by TPA has been prevented by PKC inhibitors but not by PLC inhibitors. PLC inhibitors also prevent LPS-suppressed receptor-mediated internalization of radiolabeled recombinant human (rh) M-CSF by macrophages. Similar prevention of LPS-induced M-CSF receptor downregulation was observed in human monocytes that had been pretreated with PLC inhibitors. Our results show that 1) TPA-induced M-CSF receptor loss is strictly dependent on PKC activation; 2) PLC activation alone also leads to downregulation of M-CSF receptors; and 3) LPS-induced M-CSF receptor downregulation in PEM is mediated primarily through a PLC-dependent pathway. Our data also imply that the expression of M-CSF but not GM-CSF receptors is linked to an important, yet unknown, PLC-sensitive component(s) whose hydrolysis may lead to downregulation of M-CSF receptors.

Phase I trial of recombinant macrophage colony-stimulating factor by rapid intravenous infusion in patients with cancer.

Fourteen patients were entered into a phase I dose-escalation trial of macrophage colony-stimulating factor (M-CSF). M-CSF was administered to inpatients by rapid 15 min i.v. infusion every 8 h x 5 days, repeated after a 9-day rest. Dose levels evaluated were 20, 40, 80, 330, and 1,100 micrograms/m2. Monitoring of patients every 4 h included vital signs, daily complete blood count (CBC), and serum chemistries (SGOT, creatinine, and bilirubin) while receiving M-CSF. No clinical or laboratory evidence of toxicity was seen. The average serum t1/2 varied with dose level. At 330 and 1,100 micrograms/m2, the serum t1/2 was 25 and 84 min, respectively, implying a saturable mechanism of clearance. After 5 days of treatment, the t1/2 decreased by twofold, consistent with enhancement of the saturable mechanism. Monocyte cytotoxicity against the A375 melanoma cell line was evaluated pretreatment and day 5 of each cycle. No consistent enhancement of monocyte cytotoxicity was seen. No effect on peripheral blood monocyte number was seen until the 1,100 micrograms/m2 dose level. At this dose level, the mean monocyte number on day 5 was increased compared to baseline (1,300 mm3 vs. 300/mm3). Clinical activity was seen in two patients with previously progressive leiomyosarcoma metastatic to the liver. A partial response (PR) lasting 7 months occurred at the 330 micrograms/m2 dose level while a patient treated at 1,100 micrograms/m2 has had stable disease for 20+ months. The maximum tolerated dose (MTD) of M-CSF was not determined. Based on clinical responses, a phase II trial is warranted in patients with metastatic soft tissue sarcoma.

Human lymphokine activated killer (LAK) cells suppress generation of allospecific cytotoxic T cells: implications for use of LAK cells to prevent graft-versus-host disease in allogeneic bone marrow transplantation.

We have found that murine lymphokine activated killer (LAK) cells have veto and natural suppressor activities in vitro, and prevent graft-versus-host disease (GVHD) in vivo. To determine whether human LAK cells mediate veto and natural suppression we measured their ability to inhibit generation of allospecific cytotoxic T cells (CTL) in mixed lymphocyte culture (MLC). When added to MLCs at low concentrations LAK cells caused veto-type inhibition: stimulator-type LAK cells inhibited generation of CTL but responder or third-party LAK cells did not. At higher concentrations LAK cells caused nonspecific inhibition: all three LAK cell types inhibited generation of CTL. LAK cell veto and natural suppressor activities were largely eliminated by irradiation with 30 Gy and by depletion of CD56+ cells, but increased after depletion of CD3+ cells. LAK cell veto activity is not likely an artifact of cold-target inhibition by the LAK cells themselves or by proliferation of T cells contaminating LAK cell preparations: (1) veto only occurred when LAK cells were added to MLC on days 0 through 2, but not when added on day 5; (2) addition of saturating numbers of labeled targets to fixed numbers of allo-CTL effectors failed to overcome the inhibitory effects of adding stimulator-type LAK cells at the onset of MLC; and (3) CD3-depleted LAK cells showed greater veto activity than threefold greater numbers of control LAK cells. In light of our previous findings in mice, the current results imply that adoptive immunotherapy with LAK cells may be useful in preventing GVHD in human bone marrow transplant recipients.

Sequential dacarbazine/cisplatin and interleukin-2 in metastatic melanoma: immunological effects of therapy.

Thirteen previously untreated patients with metastatic melanoma entered into a phase II chemo-immunotherapy trial were monitored immunologically during treatment. Treatment consisted of dacarbazine (DTIC) 750 mg/m2 and cisplatin 100 mg/m2 on day 1 followed by interleukin-2 (IL-2) 4 x 10(6) U/m2 by daily intravenous bolus on days 12-16 and 19-23. Cycles were repeated every 28 days. On days 1 (pretreatment), 12, 16, and 23 of each cycle, lymphokine-activated killer (LAK) cell and natural killer cell activity as well as total lymphocyte count and CD3, CD4, CD8, and CD56 lymphocyte subsets were analyzed. Despite pretreatment with full-dose cytotoxic chemotherapy, all patients were able to respond immunologically to IL-2. Spontaneous LAK cell activity was generated by the end of each course of IL-2 administration and persisted for at least 5 days thereafter. Lymphocytosis was maximum at 5 days after IL-2 administration and included increased numbers of all measured lymphocyte subsets. IL-2 administration caused a relative increase in CD56+ cells and a relative decrease in CD3+ cells. There was a direct correlation between the increase in LAK cell activity and the increase in CD56+ lymphocytes. Antitumor responses occurred in five patients but these responses did not correlate with any of the measured changes in LAK activity or lymphocyte subsets. DTIC and cisplatin administered in this schedule does not abrogate the immunological effects of IL-2.

A phase I trial of recombinant interleukin-2 combined with recombinant interferon-gamma in patients with cancer.

Twenty-six patients with metastatic cancer were entered into a phase I trial of concurrent recombinant interleukin-2 (IL-2) and recombinant interferon-gamma (IFN-gamma). IL-2 was administered as a continuous intravenous infusion for 5 days. IFN-gamma was administered by a daily intramuscular (IM) injection during the 5 days of IL-2 administration. Treatment was repeated twice after 9-day rest periods. After a 2-week rest, patients without evidence of tumor progression were retreated. Natural killer (NK)- and lymphokine-activated killer (LAK)-cell activity were assayed in each patient before treatment, on day 1, and on day 5 of each cycle. Constitutional symptoms occurred in most patients but were not dose-limiting. Other toxicities included hypotension responsive to fluids, transient elevations in liver function tests, erythema/pruritus, eosinophilia, and transient leukopenia/thrombocytopenia. The maximum-tolerated dose (MTD) of the combination was 1 x 10(6) U/m2/d of IL-2 combined with 0.50 mg/m2/d of IFN-gamma. The dose-limiting toxicity was pulmonary manifesting as rales and shortness of breath. The dose of the combination that resulted in the optimal generation of in vivo LAK-cell activity was a dose of at least 0.25 mg/m2/d of IFN-gamma combined with 1 x 10(6) U/m2/d of IL-2. Objective clinical responses were seen in five of 26 patients. These included a partial response of 2 months duration in a patient with non-Hodgkin's lymphoma (NHL), mixed responses in a patient with NHL and two patients with renal cell carcinoma (RCC), and an ongoing assessable response in a patient with bone metastases from RCC. The recommended dose for phase II trials of this combination is 0.50 mg/m2 of IFN-gamma and 1 x 10(6) U of IL-2.

Role of granulocyte/macrophage colony-stimulating factor in the regulation of murine alveolar macrophage proliferation and differentiation.

Granulocyte/macrophage (GM)-CSF is one of the hemopoietic growth factors that stimulates neutrophilic granulocyte and macrophage production by bone marrow progenitor cells. In this study, the effect of GM-CSF on the growth and differentiation of murine pulmonary alveolar macrophages (PAM) was investigated. In the presence of GM-CSF, normal murine PAM were induced to proliferate and develop into macrophage colonies with a dose-response curve similar to that of bone marrow GM colony-forming cells. PAM also responded to CSF-1, a lineage-restricted growth factor, but required much higher doses of CSF-1 and a longer incubation time for optimal colony formation. The proliferative response of PAM to CSF-1, however, was greatly enhanced by the concurrent addition of low doses of GM-CSF. In contrast, low doses of CSF-1 failed to potentiate the proliferative response of PAM to GM-CSF. Macrophages derived from GM-CSF cultures were rounder and less stretched and possessed less FcR-mediated phagocytic activity than cells produced in CSF-1 cultures. A study with hydrocortisone-induced monocytopenia showed that nearly one half of lung macrophages may be sustained by local proliferation of PAM without the continuous migration of blood monocytes. This study suggests that GM-CSF may play a major role in the production of PAM by two modes of action, 1) direct stimulation of cell proliferation and 2) enhancement of their responsiveness to CSF-1, thereby producing more mature and functionally competent macrophages.

Granulocyte/macrophage colony-stimulating factor stimulates monocyte and tissue macrophage proliferation and enhances their responsiveness to macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a specific humoral growth factor that stimulates both neutrophilic granulocyte and macrophage production by bone marrow hematopoietic progenitor cells. GM-CSF also stimulates the proliferation and clonal growth of both tissue macrophages and blood monocytes. Although at low concentrations GM-CSF was unable to support the long-term growth of tissue macrophages, it greatly enhanced their responsiveness to macrophage CSF (M-CSF, or CSF-1). This effect was also observed by treating macrophages with GM-CSF for a short time. GM-CSF did not compete with M-CSF for binding to M-CSF receptors nor was it inactivated by treatment with anti-M-CSF antiserum. Treatment of tissue macrophages with GM-CSF led to a rapid but transient downregulation of M-CSF receptors; prolonged incubation at 37 degrees C, however, resulted in a restoration and upregulation of M-CSF receptors. Identical effects were observed with both native or recombinant GM-CSF. This study suggests that GM-CSF regulates tissue macrophage production by two modes of action: (a) direct stimulation of macrophage proliferation, and (b) enhancement of their responsiveness to M-CSF.

Delineation of receptor-mediated colony-stimulating factor (CSF-1) utilization and cell production by precursors of mononuclear phagocytic series at various stages of differentiation.

Colony-stimulating factor-1 (CSF-1) is a specific haematopoietic growth factor that stimulates the production of macrophages by both bone marrow macrophage precursors (GM-CFC) and certain more mature peripheral tissue macrophages. The relationship of CSF-1 utilization and cell production by macrophage precursors at various stages of differentiation was studied. Bone marrow GM-CFC had the highest proliferative capacity followed by blood monocytes and peritoneal exudate macrophages (PEM) as determined by their cell doubling time (DT) which was also dependent on the concentrations of exogenous CSF-1. PEM had the longest initial lag period before commencing cell proliferation. Exogenous CSF-1 was constantly utilized by the growing cells; depletion of available CSF-1 resulted in growth arrest and, subsequently, cell death. The production of macrophage progeny, per amount of CSF-1, correlated with parent macrophage maturity; for each 100 U of CSF-1 consumed, bone marrow precursor cells and blood monocytes were capable of producing 17.9 x 10(4) and 13.4 x 10(4) progeny, respectively, whereas PEM generated only 4.6 x 10(4) daughter cells. Thus, the removal and destruction of CSF-1 by more mature, less proliferative tissue macrophages may provide a possible mechanism by which CSF-1 levels are reduced and the production of early haemopoietic macrophage precursors controlled.

Expression of gamma-interferon receptor in murine bone marrow-derived macrophages associated with macrophage differentiation: evidence of gamma-interferon receptors in the regulation of macrophage proliferation.

The effect of purified, recombinant murine gamma interferon (IFN-gamma) on the regulation of macrophage proliferation induced by colony-stimulating factor 1 (CSF-1) was investigated. Although both hemopoietic stem cells (GM-CFC) and tissue-derived peritoneal exudate macrophages (PEM) proliferated in response to CSF-1, the more mature PEM were much more sensitive to an antiproliferative effect of IFN-gamma. The role of IFN-gamma receptor expression and its relationship to growth inhibition was examined. Bone marrow cells as a whole did not exhibit an appreciable amount of IFN-gamma receptor binding activity. Likewise, nonadherent (NA) cells derived from CSF-1-stimulated bone marrow cultures displayed low levels of IFN-gamma receptor binding activity. On the contrary, more mature adherent (AD) cells (monocytes/macrophages) from the same culture exhibited high levels of IFN-gamma receptor binding activity, which continued to increase with culture time. The elevated IFN-gamma binding activity is due to an increase in total receptor number rather than the binding affinity as judged by Scatchard analysis. Similar to the relationship between PEM and GM-CFC, more mature AD cells were also more susceptible to the inhibitory effect of IFN-gamma on CSF-1-induced proliferation than their less mature NA counterparts. The fact that the sensitivity to IFN-gamma correlated well with the expression of existing IFN-gamma receptors strongly suggests that the inhibitory effect is mediated through IFN-gamma receptors. This study shows that the expression of IFN-gamma receptors in mononuclear phagocytes may not only represent one of the phenotypic parameters acquired by the growing macrophages during the process of differentiation, but may play some role in controlling proliferation.

Induction of prostaglandin production by hyperthermia in murine peritoneal exudate macrophages.

In response to inflammatory stimuli, macrophages synthesize and secrete prostaglandins (PGE) along with other potent inflammatory mediators. We have studied the effect of hyperthermia on the production of PGE by murine mononuclear phagocytes. Exposure to high temperature induced PGE production by cultured C3H/HeJ exudate macrophages in a time- and temperature-dependent manner. Increase in PGE production was detected when macrophages were treated at 41 degrees C and above for 1 h with a much greater increase at 42 and 43 degrees C. The secretion of PGE into culture supernatants by heat-treated macrophages reached a maximum approximately 24 h after heat treatment. The production of PGE by macrophages after hyperthermia was inhibited either by the addition of 5 X 10(-7) M indomethacin or by the subsequent incubation at 4 degrees C, suggesting that the elevated PGE production by macrophages is mediated through the activation of cyclooxygenase. Heat treatment under the same conditions failed to stimulate the production of PGE by either a human monocyte-like tumor cell line (U-937) or a mouse fibroblast cell line (L-929).

Tumor-localizing components of the porphyrin preparation hematoporphyrin derivative.

Synthetic and analytical approaches were used to characterize the tumor-localizing components of the porphyrin preparation, hematoporphyrin derivative. From studies involving aqueous and nonaqueous gel exclusion and reverse-phase chromatography, we conclude that localization is mediated by hematoporphyrin derivative components which are among the most hydrophobic in the preparation. This apparent hydrophobicity may derive from hydrogen-bonding phenomena, rather than from absence of hydrophilic functional groups.