Allergic airway hyperreactivity increases the risk for corneal allograft rejection.

Corneal allografts transplanted into hosts with allergic conjunctivitis experience an increased incidence and swifter tempo of immune rejection compared to corneal allografts transplanted to nonallergic hosts. Previous findings suggested that increased risk for rejection was not a local effect produced by an inflamed eye, but was due to perturbation of the systemic immune responses to alloantigens on the corneal allograft. We tested the hypothesis that another allergic disease, airway hyperreactivity (AHR), would also increase the risk for corneal allograft rejection. Induction of AHR with either ovalbumin (OVA) or short ragweed (SRW) extract prior to keratoplasty resulted in a steep increase in the speed and incidence of corneal allograft rejection. Delayed-type hypersensitivity (DTH) responses to corneal alloantigens were closely associated with corneal allograft rejection. However, the deleterious effect of AHR on corneal allograft survival was not reflected in a heightened magnitude of allospecific DTH, cytotoxic T lymphocyte and lymphoproliferative responses to the alloantigens on the corneal allograft. Unlike Th2-based immediate hypersensitivity, CD8+ T-cell-based contact hypersensitivity to oxazolone did not increase the risk for corneal allograft rejection. Thus, Th2-based allergic diseases significantly reduce the immune privilege of the corneal allograft and represent important risk factors for consideration in the atopic patient.

Cytochalasin B prevents specific sorting of reaggregating embryonic cells.

The effect of cytochalasin B on specific sorting during reaggregation of embryonic chick heart and neural retina cells was studied. At a dose that did not measurably affect uptake of precursors of protein and RNA synthesis, ratios of potassium to sodium ions, and nonspecific aggregation, cytochalasin B disrupted the formation of the characteristic pattern of islands of heart cells within a retinal continuum.

Flight Lieutenant Peach's observations on Burning Feet Syndrome in Far Eastern Prisoners of War 1942-45.

INTRODUCTION: 'Burning Feet Syndrome' affected up to one third of Far Eastern Prisoners of War in World War 2. Recently discovered medical records, produced by RAF Medical Officer Nowell Peach whilst in captivity, are the first to detail neurological examinations of patients with this condition. METHODS: The 54 sets of case notes produced at the time were analysed using modern diagnostic criteria to determine if the syndrome can be retrospectively classed as neuropathic pain. RESULTS: With a history of severe malnutrition raising the possibility of a peripheral polyneuropathy, and a neuroanatomically plausible pain distribution, this analysis showed that Burning Feet Syndrome can now be described as a 'possible' neuropathic pain syndrome. CONCLUSION: After 70 years, the data painstakingly gathered under the worst of circumstances have proved to be of interest and value in modern diagnostics of neuropathic pain.

Inhibition of embryonic cell aggregation by neoplastic cells.

The effects of normal and malignant cells on the aggregation of embryonic cells in gyratory shaker cultures were compared. The addition of 1 times 10-5 simian virus 40 (SV40)-transformed BALB/3T3 (SV40-3T3) cells to 6 times 10-6 embryonic neural retina cells caused a highly significant greater reduct on (22.7 percent) in aggregate diameter than the addition of untransformed BALB/3T3 (3T3) cells. The ratio of the number of single cells to the number of aggregates was significantly higher for cultures containing SV40-3T3 cells than for the cultures containing 3T3 cells. This effect was concentration dependent in the presence of cultured Ehrlich-Lettre hyperdiploid (ELD) ascites cells; however, media from ELD cell cultures or ELD cell sonicates resulted in aggregates of greater diameter and lower ratios of single cells to aggregates. This approach may provide a sensitive assay system for the interactions of tumor and other cells in vitro.

Differential inhibition of in vitro malignant cell assays by B16 mouse melanoma variants.

Neural retina aggregation and adhesion assays, shown to be sensitive to the presence of small number of malignant cells, were differentially inhibited by cells or medium from B16 mouse melanoma sublines with higher metastatic characterizations, which suggests a correlation between inhibition of adhesion and aggregation and metastatic potential.

Some effects of the tumor promoter 12-0-tetradecanoylphorbol 13-acetate on cell interactions in vitro.

Studies were made of the effects of the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), 4-O-methyl TPA (4-O-MeTPA), and 4 alpha phorbol 12,13-didecanoate (4 alpha PDD) on the aggregation of embryonic chick cells in gyratory shaker culture, a model system useful for the study of cell adhesion and cell interactions. TPA and, to a lesser extent, 4-O-MeTPA significantly reduced the neural retina aggregate size at concentrations as low as 10(-9) M and 10(-8) M, respectively. An inactive isomer, 4 alpha PDD, had no effect up to 10(-6) M. The reduction in aggregate size appeared related to promoter activity since dexamethasone, a steroid that inhibits tumor promotion by TPA, significantly reversed the inhibitory effect of TPA. None of the agents tested affected the sorting pattern in mixed neural retina and heart cultures. The results indicate that intercellular adhesion, as determined by extent of aggregation, is reduced in the presence of TPA. This inhibition is considered to be related to the tumor-promoting activity of TPA.

Effects of liposome-entrapped doxorubicin on liver metastases of mouse colon carcinomas 26 and 38.

The effects of doxorubicin (DOX) and DOX entrapped in standardized liposomes [mean diameter, 0.15 micron; (DOX-Lip)] on the survival of mice bearing liver metastases of mouse colon carcinoma CT38LD (C57BL/6J mice) or CT26 (BALB/c mice) were investigated. In vitro cultured CT38LD cells were more sensitive to DOX than CT26. In vivo DOX and DOX-Lip, administered iv 10 mg/kg weekly to a maximum of five injections, increased the life-spans of mice bearing CT38LD liver metastases 32% (P less than .05) and 64% (P less than .05), respectively. DOX-Lip was more effective than DOX in prolonging survival (P less than .05). Free DOX did not significantly increase the life-spans of mice bearing CT26 liver metastases (P greater than .5), whereas DOX-Lip increased the life-spans 35% (P less than .05). The results suggest that liposomal delivery of agents to the liver can enhance therapeutic activity and could be used as an arm of protocols for adjuvant therapy of liver metastases.

Therapy of central nervous system leukemia in mice by liposome-entrapped 1-beta-D-arabinofuranosylcytosine.

Studies were undertaken to determine the therapeutic effects of liposome-encapsulated 1-beta-D-arabinofuranosylcytosine (lip-ara-C) against intracranial L1210 leukemia. The effects of administration route, drug dosage, liposome type, and tumor load on therapeutic efficacy were also studied. One hundred % mice were cured after a single intracranial 40 mg/kg dose of lip-ara-C, dependent on tumor load. Intracranial lip-ara-C was more effective than i.v. lip-ara-C. A single i.v. dose of lip-ara-C was therapeutically superior to 5-day i.v. infusion of the free drug. Intracranial or i.v. lip-ara-C at therapeutic doses resulted in less systemic toxicity than i.v. infusion of free ara-C, suggesting possible use of lip-ara-C as an adjunct to treatment of central nervous system leukemia.

Alterations in murine host defense functions by adriamycin or liposome-encapsulated adriamycin.

Peritoneal exudate cells (PEC) from C57BL/6 mice were collected on different days following an i.p. injection of Adriamycin (10 mg/kg) as free drug (ADM) or encapsulated in multilamellar liposomes (ADM/Lip). Macrophages harvested from mice at various times (Days 4-14) after either drug treatment were responsive to in vitro lipopolysaccharide induction of tumoricidal activity, maximum response being seen on Day 7. In addition, 18 days after treatment, significant macrophage tumoricidal activity was observed only in the ADM/Lip-treated group. When supernatants from cultures of PEC obtained 7 days after treatment were assayed for interleukin 1 following lipopolysaccharide stimulation, activity was found with both ADM- and ADM/Lip-treated cells. Without lipopolysaccharide stimulation, only PEC from ADM-treated mice elaborated factor(s) with interleukin 1-like activity. Both ADM and ADM/Lip induced significant PEC-natural killer (PEC-NK) activity by Day 4, while the ADM/Lip treatment sustained PEC-NK activity more effectively than free drug at later time points (7 or 11 days posttreatment). Drug-induced PEC-NK activity (Day 7) was (a) ablated by treatment in vitro with anti-asialo GM1 antibody and complement, and (b) associated with a population of PEC nonadherent to plastic. A transient suppression of splenic NK activity was seen 4 days following either ADM or ADM/Lip administration with recovery to control level by Day 7. These data demonstrate that following ADM or ADM/Lip administration some of the changes necessary for macrophage tumoricidal activation must have occurred in vivo. Liposome encapsulation of ADM extended the duration of ADM-induced augmentation of certain host defenses.

Antitumor effect of taxol-containing liposomes in a taxol-resistant murine tumor model.

Taxol is a promising agent for use in ovarian cancer and other malignancies. One problem associated with taxol is its low aqueous solubility, requiring Cremophor EL (polyethoxylated castor oil) and ethanol as excipients (Diluent 12); these agents cause serious adverse effects. Liposomes containing taxol and phospholipid (in a 1:33 mole ratio, respectively) were prepared from phosphatidylglycerol and phosphatidylcholine in a 1:9 mole ratio. Antitumor effect was evaluated against Colon-26, a taxol-resistant murine tumor. Given as 1, 4, or 9 injections, free taxol given i.v. in Diluent 12 was ineffective at delaying tumor growth at doses < or = 30 mg/kg per injection (the maximum tolerated dose). In contrast, taxol-liposomes were well tolerated at doses greater than or equal to the maximum tolerated dose of free taxol and showed significant tumor growth inhibition at 10-45 mg/kg per injection.

Inhibition of tumor cell growth in vitro and in vivo by 1-beta-D-arabinofuranosylcytosine entrapped within phospholipid vesicles.

Phospholipid vesicles have been used as a carrier vehicle to enhance the cytotoxic activity of 1-beta-D-arabinofuranosyl-cytosine (ara-C) and 1-beta-D-arabinofuranosylcytosine 5'-triphosphate against several tumor cell lines. The activity of both compounds in free solution or entrapped within phospholipid vesicles was compared against L1210 cells, Ehrlich ascites cells, and SV40-transformed 3T3 cells in vitro. In addition, the activity of vesicle-entrapped ara-C against L1210 cells was also studied in vivo. The results obtained in vitro with ara-C indicated no difference in the concentration needed to inhibit growth of cells by 50% between free ara-C and vesicle-entrapped ara-C. In contrast, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate entrapped in phospholipid vesicles was a more potent inhibitor of L1210 in culture (ID50, 2 X 10(-8) M) compared to the relatively inactive free 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (id50 greater than 10(-7) M). Experiments carried out with L1210 cells in mice showed that, after a single i.p. dose (10 mg/kg) of vesicle-entrapped ara-C, the average survival times of mice inoculated with 10(5) L1210 cells were increased by over 90%. In control experiments, free ara-C or vesicles plus free ara-C (10 mg/kg) did not prolong survival of mice.

Differential inhibition of embryonic cell aggregation by cultured human cells with "malignant" of "normal" characteristics.

The effect that cultured human cells have on chick embryonic neural retina cell aggregation was examined. Different types of human cultured cells inhibited aggregation of chick neural retinal cells to differetn degrees when mixed at a human cell:retina cell ration of 1:60. It appeared from the eleven cell lines studied that cells with "malignant" characteristics inhibited retinal cell aggregation to a greater extent than those with more "normal" characteristics. The assay could be used as a further test for abnormality of cell types and also as a method for studying the interactions of malignant cells with cultured cells.

Effects of partially thiolated polycytidylic acid and liposomes on in vitro colony-forming cells of leukemic mice.

Partially thiolated polycytidylic acid (MPC), an antileukemic agent, when administered to leukemic RF/UN mice inhibited the clonogenicity of bone marrow progenitor cells in a time- and dose-dependent manner. The effect of a single dose of MPC disappeared within 40 hr due to the rapid degradation of this compound in mice. When MPC was encapsulated in liposomes before injection, its activity at 19 hr after inoculation was similar to that of free MPC. The inhibitory effect of this liposome-MPC complex, however, persisted for at least 40 hr, indicating that the MPC was protected from hydrolysis by the nucleases present in blood. Drug-free liposomes increased the number of clonogenic progenitor cells, whereas a mixture of plain liposomes and MPC decreased the number of clonogenic cells to a greater extent than did MPC alone or MPC within liposomes. A possible explantation for these observations is that the liposomes per se altered the clearance function of the reticuloendothelial system and completed with MPC for uptake by the reticuloendothelial system cells, thereby resulting in increased plasma levels of MPC which in turn resulted in greater killing of the target cells.

Arrest of human lung tumor xenograft growth in severe combined immunodeficient mice using doxorubicin encapsulated in sterically stabilized liposomes.

Incorporation of polyethylene glycol-derivatized phospholipids into liposomes results in carriers that can enhance the therapeutic efficacy of encapsulated drugs by imparting the ability to evade the reticuloendothelial system and remain in the circulation for prolonged periods. In this study, doxorubicin encapsulated in these sterically stabilized liposomes (S-DOX) is shown to completely arrest the growth of human lung tumor xenografts in severe combined immunodeficient (scid) mice. Doxorubicin administered at equivalent doses as free drug or encapsulated into conventional liposomes was ineffective at completely arresting the growth of this human tumor, although a decrease in tumor growth rate compared to untreated controls was observed. Scid mice were found to be significantly more susceptible to the toxic effects of doxorubicin than were immunocompetent C.B-17 control mice, a characteristic that is likely to result from the deficit in DNA repair mechanisms previously identified in scid mice. However, doxorubicin toxicity in scid mice could be minimized while maintaining the antitumor activity of doxorubicin encapsulated in sterically stabilized liposomes by administering the drug in multiple weekly injections at low doses. This report provides the first evidence that antitumor drugs delivered in sterically stabilized liposomes are more effective at arresting the growth of human tumors than are conventional delivery systems. In addition, the scid mouse is presented as a viable model in which to study novel chemotherapeutic approaches to the treatment of human cancer.

Pharmacokinetics and therapeutics of sterically stabilized liposomes in mice bearing C-26 colon carcinoma.

Three different liposome types were compared for blood clearance and tissue uptake in mice bearing C-26 colon carcinoma growing either s.c. or in liver. Therapeutic experiments were performed with the liposome preparation showing the highest tumor uptake. Liposomes were composed of solid-phase phosphatidylcholine, either distearoyl phosphatidylcholine or hydrogenated soy phosphatidylcholine, and cholesterol at a 2:1 molar ratio. These liposomes were compared with similar but sterically stabilized liposomes (SL) which, in addition, contained either GM1 ganglioside or phosphatidylethanolamine derivatized with poly(ethylene glycol). Pharmacokinetic analysis of drug disposition was based on the areas under the curve for liposome-entrapped 67Ga uptake per gram of tissue up to 96 h following i.v. injection. The highest tissue area under the curve values with both liposome types were obtained in spleen, liver, and tumor. However, the sterically stabilized liposomes gave an area under the curve value 2-3-fold higher in the s.c. tumor and about 2-fold lower in liver and spleen. The therapeutic efficacy of doxorubicin (DOX) and epirubicin (EPI) encapsulated in poly(ethylene glycol)-derivatized phosphatidylethanolamine-containing liposomes was compared with that of free drug at two doses, 6 and 9 (or 10) mg/kg animal weight. Liposomes containing drug were injected either as a single dose, at different times following tumor implantation, or as three weekly doses starting 10 days after implantation. When injected as a single dose, liposome-encapsulated DOX had the maximal effect on tumor growth when injected 6 to 9 days after tumor implantation. When injected as three weekly doses, with treatment starting with a delay of 10 days, tumors which had grown to a size of approximately 0.05-0.1 cm3 regressed in groups of animals treated with either liposome-encapsulated drug (SL-DOX or SL-EPI) but continued to grow unabated in untreated mice and in mice receiving either of the free drugs. Survival of tumor-bearing animals treated with either SL-EPI or SL-DOX was significantly prolonged. Animals receiving saline, EPI, or DOX survived a mean of 50, 62, and 49 days, respectively, following tumor implantation. Eight of nine and nine of 10 animals receiving 6 and 9 mg/kg SL-EPI, respectively, survived to 120 days. Ten of 10 animals in both groups receiving 6 and 9 mg/kg SL-DOX survived to 120 days. None of the surviving animals in the SL-EPI and SL-DOX group showed any histological evidence of tumor at the conclusion of the experiment (120 days).(ABSTRACT TRUNCATED AT 400 WORDS)

Immunospecific targeting of cytosine arabinonucleoside-containing liposomes to the idiotype on the surface of a murine B-cell tumor in vitro and in vivo.

A new tumor model is described that is suitable for the evaluation of antibody-directed drug-delivery protocols and a modification in the procedure for covalently coupling antibody to the surface of drug-containing liposomes is presented. These immunospecific liposomes containing cytosine arabinonucleoside (Ara-C) have been tested in vitro and in vivo for their ability to kill a B-cell tumor. The target of the immunospecific-Ara-C liposomes is the idiotype associated with an antigen-specific immunoglobulin receptor on the cell surface of a murine B-cell hybrid (2C3). Affinity-purified antibodies specific for the idiotype were covalently coupled to modified lipid on the surface of the large unilamelar liposomes containing drug. These liposomes were shown to kill idiotype-positive 2C3 cells in vitro, but not idiotype-negative variants of this same cell line. It was also established in vitro that the drug-containing liposomes were at least 40 times more efficient than free Ara-C in the killing of the tumor cells. The 2C3 tumor was also propagated in vivo following the i.p. administration of tumor cells. The tumor grew initially as multiple foci within the peritoneum and subsequently spread to the spleen. Tumor-bearing mice were treated either with free Ara-C or with immunospecific liposomes containing Ara-C. Tumor growth in the primary tumor nodules and in the spleen was monitored by the administration of bromodeoxyuridine to the tumor-bearing animals followed by the immunofluorescent staining of cells with a monoclonal anti-bromodeoxyuridine antibody to estimate the proportion of cells in S phase. Our data from five out of seven animal experiments shows that the immunospecific-Ara-C liposomes, but not free drug, reduced tumor growth in the spleen. However, neither the liposomes containing drug nor the free drug were able to alter the growth of the primary tumor nodules growing in the peritoneal cavity. These results suggest that immunospecific-Ara-C containing liposomes may be useful in conjunction with other cytoreductive protocols in controlling tumor growth or preventing the spread of the tumor to other sites, but that immunospecific-Ara-C containing liposomes by themselves are not likely to eliminate an established tumor in vivo. We also demonstrate here that the administration of immunospecific-Ara-C containing liposomes in an animal having high levels of circulating tumor-associated antigen (i.e., IgG containing the idiotype) represents a potential clinically relevant hazard which must be considered when designing antibody-directed drug-delivery protocols.

Enhanced therapeutic effects of liposome-associated 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine.

The ether-lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) has anticancer activity, but systemic toxicity has restricted its therapeutic use. In this report "free" ET-18-OCH3 and a stable, well-characterized, liposome-based formulation of ET-18-OCH3 (ELL-12) were compared for in vivo toxicity in normal mice and for therapeutic efficacy in three mouse tumor model systems. The entrapment of ET-18-OCH3 in liposomes decreased the acute toxicity of ET-18-OCH3 after i.v. administration. The maximum tolerated dose for a single i.v. dose of free ET-18-OCH3 was found to be approximately 25 mg/kg, whereas the maximum tolerated dose for ELL-12 was approximately 200 mg/kg. ELL-12 was much less hemolytic in vivo than ET-18-OCH3. The therapeutic efficacy of free ET-18-OCH3 and ELL-12 was investigated against i.p. P388 leukemia, Lewis lung cancer lung metastases, and B16/F10 melanoma (lung tumor nodules) in mice. Although ET-18-OCH3 had some anticancer activity, it was found that ELL-12 was more effective than ET-18-OCH3 in all three tumor models at lower and nontoxic dose schedules. These results suggest that association of ET-18-OCH3 in stable, well-characterized liposomes transforms it into an effective antitumor agent.

Cell growth and quabain-sensitive 86Rg+ uptake and (Na++K+)-ATPase activity in 3T3 and SV40 transformed 3T3 fibroblasts.

The uptake of ouabain-sensitive 86Rb+ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive 86Rb+ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive 86Rb+ uptake in both cell types. This increase ouabain-sensitive cation transport was reflected in increased levels of (Na++K)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same Km as 3T3 cells. These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier.

Cultured Ehrlich ascites tumor cells equilibrate D-glucose via a carrier mechanism with a Km and V of 14 mM and 3 mu mol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (Ki) of approx. 5.10(-7) M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1.10(-5) M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (Kd) of approx. 1.10(-6) M, represents about 30% of the total cytochalasin B binding of the cell (8.10(6) molecules/cell), is sensitively displaced by cytochalasin E but not by D-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a Kd of 4--6.10(-7) M, represents approx. 60% of the total saturable binding (14.10(6) molecules/cell), is specifically displaced by D-glucose with a displacement constant of 15 mM, but not by L-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a Kd of 2--6.10(-8) M, represents less than 10% of the total sites (2.10(6) molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.

Reversible depression of the reticuloendothelial system by liposomes.

The effect of various doses of different types (reverse phase evaporation vesicles and small unilamellar vesicles) of intravenously injected liposomes on reticuloendothelial activity, as measured by the blood clearance rate of intravenously injected carbon, was investigated. Also the effect of pretreatment with reverse phase evaporation vesicles on blood clearance and tissue distribution of a second dose of similar vesicles was determined. For all concentrations used reverse phase evaporation vesicles caused a reduction in reticuloendothelial activity at least up to 4 h after injection. 24 h after administration the rate of carbon clearance returned to the control level. On the contrary small unilamellar vesicles did not block reticuloendothelial activity. Pretreatment with reverse phase evaporation vesicles (250 mumol/kg) caused an increased blood level and a decreased hepatic uptake of a second dose of the vesicles, injected 1 h after the first dose. This seems to be due to a depression of reticuloendothelial activity and not to a depletion of opsonins. Pretreatment with small unilamellar vesicles (250 mumol/kg) had no significant influence on the tissue distribution of a second dose of vesicles. Our results clearly indicate that reverse phase evaporation vesicles cause a reversible depression of reticuloendothelial activity and this depression seems to be induced by a saturation of reticuloendothelial cells with liposomes.