Differentiation therapy of a myelomonocytic leukemia (c-WRT-7) in rats by injection of lipopolysaccharide and daunomycin

We examined the therapeutic effect of lipopolysaccharide (LPS), a differentiation inducer, in combination with daunomycin, an antileukemia drug possessing differentiation-inducing potential, on a rat myelomonocytic leukemia (c-WRT-7). c-WRT-7 cells were found to differentiate into macrophage-like cells and to lose their growth capacity both in vitro and in vivo after incubation with LPS. Morphological differentiation of c-WRT-7 cells was observed in diffusion chambers which had been inserted into the abdominal cavity of syngeneic WKA rats given injections of LPS. A series of i.p. injections of LPS resulted in the complete inhibition of the leukemia development in about 60% of the rats while the remaining rats showed a significant prolongation of survival time when they were given i.p. injections of LPS-sensitive c-WRT-7 cells. The effect of LPS was minimal, however, in those rats which had been given i.p. injections of LPS-hyporesponsive c-WRT-7 cells. Although an i.v. injection of 100 untreated c-WRT-7 cells was enough to kill the syngeneic rats, combination treatment with LPS and daunomycin was able to inhibit completely the development of leukemia in those rats which had been given i.v. injections of LPS-sensitive c-WRT-7 cells, whereas the same treatments were only partially effective in the prolongation of survival among those rats which had been given i.v. injections of LPS-hyporesponsive c-WRT-7 cells. Our studies show that LPS was capable of curing a proportion of rats and that it significantly prolonged the survival of the remainder who had received a transplantation of the differentiation-sensitive leukemia cells; this action was significantly enhanced by the associated administration of daunomycin.     

Local injection of OK432 can augment the TH1-type T-cell response in tumor-draining lymph node cells and increase their immunotherapeutical potential

The effect of local injections with streptococcal preparation OK432 on the therapeutical potential of tumor-draining lymph node (LN) cells was investigated in mice. Peritumoral injections with OK432 on days 2, 4, 6, 8 and 10 showed no effect on the in vivo growth of s.c. inoculated B16F10 melanoma. The B16F10-draining OK432-treated LN cells, however, showed a high level of anti-B16F10 cytolytic activity after an in vitro culture first with both anti-CD3 monoclonal antibody (MAb) and activated B cell blasts, and subsequently with interleukin (IL)-2 without in vitro restimulation. Such in vitro expanded LN cells showed a remarkable antitumor effect against pulmonary metastasis of B16F10 melanoma, even without the concurrent administration of IL-2. In addition, the therapeutical protocol was also found to be moderately effective against poorly immunogenic MCA fibrosarcoma, and the in vivo antitumor effect was specific to the tumor from which the LNs were harvested. Interestingly, 2 kinds of comparative analyses of the cytokines revealed that the B16F10-bearing state induced the draining LN cells to develop a Th2-type response. However, the OK432 treatment was able to effectively augment their Th1-type response. Collectively, our results suggest that peritumoral injections with OK432 significantly increased the therapeutical potential of the tumor-draining LN cells by augmenting their Th1-type response.Int. J. Cancer 70:606–611. © 1997 Wiley-Liss Inc.     

Uptake of radiolabeled alpha-fetoprotein by mouse mammary carcinomas and its usefulness in tumor scintigraphy

The ability to internalize alpha-fetoprotein (AFP) and serum albumin, which is characteristic of embryonic and fetal elements undergoing differentiation, may reappear in some cultured neoplastic cells (i.e., the MCF-7 human breast cancer cell line). The in vivo uptake of AFP by spontaneous carcinomas of the CH3/Bi mouse was investigated. Nineteen mice were given i.v. injections of approximately 10 muCi of mouse 125I-AFP (0.6 to 4 micrograms of AFP according to the specific ratio of the preparation used). Four to 7 days later, the animals were sacrificed. The radioactivity concentration in the tumor was the highest among all solid tissues examined. Tumor:liver radioactivity ratios were clearly positive [3.6 +/- 0.3 (S.E.)] in 27 of 31 specimens studied. Microscopy examination of autoradiograms from various tissue sections confirmed the selective accumulation of radioactive AFP in the tumors. In order to assess the specificity of AFP uptake by mammary tumors, 4 mice were given simultaneous injections of 125I-AFP and 131I-ovalbumin, respectively. Compared to AFP, the retention of ovalbumin was very low in all tissues studied, including the tumor. The possibility of tumor localization of radiolabeled AFP by external photoscanning was also explored. Two mice were given injections of 131I-AFP, one mouse received 131I-serum albumin, and one was given 131I-ovalbumin. Images were obtained 6 days after with a standard gamma-camera linked to a computer with data display. About 50% of the total radioactivity retained was concentrated in the tumor areas of mice given injections of iodinated AFP, while it was only 15% in the mouse that received 131I-serum albumin. No tumor image could be detected in the mouse given ovalbumin. These results show that the ability to internalize AFP, common to many tissues during ontogenesis, may also be shared by neoplastic cells which develop later in life. They also prove the preferential uptake of AFP by the tumors compared to normal tissues and the usefulness of AFP as a radiotracer for mammary carcinomas. The latter represents a novel approach to tumor detection.     

华蟾素注射液对人肝癌HepG-2细胞DNA拓扑异构酶Ⅰ的影响

背景与目的:华蟾素注射液是传统抗肿瘤中药制剂,目前药效机制尚不明确.本研究旨在探讨华蟾素注射液对DNA拓扑异构酶Ⅰ(topoisomerase Ⅰ,TOPO Ⅰ)的影响.方法:采用噻唑蓝还原法(MTT)观察华蟾素注射液对人肝癌HepG-2细胞增殖的影响;采用流式细胞术(FCM)检测对HepG-2细胞周期的影响;采用RT-PCR法检测对HepG-2细胞TOPO Ⅰ mRNA表达的影响;采用TOPO Ⅰ介导的负超螺旋PBR322 DNA解旋反应检测华蟾素对TOPO Ⅰ酶活性的影响;采用负超螺旋PBR322 DNA检测华蟾素对DNA的直接抑制作用.结果:华蟾素注射液对HepG-2细胞增殖具有抑制作用,其抑制效应具有时间和剂量依赖性;并将HepG-2细胞阻滞于S期;RT-PCR检测表明,华蟾素注射液可下调TOPO Ⅰ mRNA表达,表现浓度依赖效应;对TOPO Ⅰ介导的负超螺旋PBR322 DNA解旋反应有抑制作用:对负超螺旋PBR322 DNA没有直接抑制作用.结论:华蟾素注射液能够抑制HepG-2细胞增殖,影响DNA拓扑异构酶Ⅰ酶的mRNA表达及活性可能为机制之一.     

Development of an in vitro assay for the survival of cells suspended from BA1112 rat sarcomas

A method has been developed for the in vitro assay of the survival of BA1112 rat sarcoma cells following treatment in vivo. The BA1112 tumor was not adapted for growth in culture and was maintained by serial transplantation in vivo. The assay used a feeder layer of heavily irradiated cells to eliminate any changes in plating efficiency caused by the large number of dying cells which must be plated when assaying cell survival after intensive treatments. The plating efficiency of the tumor cells was not affected significantly by the age of the feeder layer (between 1 and 4 days), the density at which the experimental cells were plated or the interval between preparation of the cell suspension and plating (up to 4 hr). Using the in vitro assay a survival curve was determined for cells from tumors irradiated in air-breathing animals. This curve was similar to that determined previously for BA1112 tumor cells using an in vivo assay to measure cell survival.     

Effect of syngeneic tumor cells bound to concanavalin A on tumor growth

Transplanted MOPC 315 solid myelomas in BALB/cCR mice were treated with single injections of 1.6 × 10⁶ syngeneic MOPC 315 cells combined either with human gamma globulin or with Concanavalin A (Con A). The group of mice which received injections of MOPC 315 cells coupled with human gamma globulin failed to show any significant response. The second group which received injections of MOPC 315 cells coated with Con A responded in variable fashion. Response was measured in terms of delay of tumor growth, differences in growth pattern, and prolongation of survival time. Slight response was observed in 8 of the 20 mice; moderate response was seen in 6 mice; 6 mice did not respond. The data obtained point to a possibly effective tumor vaccine which deserves further study.     

Immunotherapy of a mouse mammary carcinoma by sustained peritumor release of IL-2

The purpose of this study was to compare the local and systemic therapeutic effects of Interleukin-2 (IL-2) used in 3 different preparations: suspended in PBS, suspended in 2% agar, and entrapped in multi-lamellar liposomes suspended in 2% agar. The liposomes were composed of phosphatidylglycerol and phosphatidylcholine in a 1:4 molar ratio. The net release of IL-2 in vitro (by ELISA assay) at 37 degrees C, measured at 4 hr, 2 days, and 10 days, was 50%, 75%, and 100% from agar, and 8%, 22%, and 33% from liposomes in agar. In the therapeutic tests, the IL-2 preparations were injected close to s.c. implants of the MC2 mouse mammary carcinoma. Four injections at weekly intervals of IL-2 in agar had as much local and systemic (against uninjected contralateral tumor implants in treated mice) therapeutic effect as the same total amount of IL-2 in PBS given in 20 daily injections over 4 weeks. The IL-2 liposome-gel preparation was most effective (p less than 0.05), probably due to the more sustained release of IL-2. Three injections of this preparation gave a fixed and sustained peritumor release of IL-2 which, at sub-toxic levels, resulted in both local and systemic therapeutic effects.     

Immunotherapy of established micrometastases with Bacillus Calmette-Guérin tumor cell vaccine.

We evaluated the use of Bacillus Calmette-Guérin admixed with tumor cells as a vaccine to induce systemic tumor immunity for therapy of subclinical (micrometastatic) disease. In several experiments inbred strain 2 guinea pigs were given i.v. injections of either 10(4), 10(5), or 10(6) syngeneic L10 hepatocarcinoma cells, and initial vaccinations were administered either 1 or 4 days after tumor inocluation. Variables in vaccine preparation, such as ratio of viable Bacillus Calmette-Guérin organisms to tumor cells, procedures for freezing the tumor cells, X-ray treatment of tumor cells, and vaccination regimen were evaluated. The studies demonstrated that under defined conditions nontumorigenic vaccines of Bacillus Calmette-Guérin and tumor cells can cure the majority of animals of otherwise lethal visceral micrometastases.     

Studies on estrogen induced pituitary tumor in the rat with special reference to the relationship of the tuberoinfundibular dopamine neuron system

Pituitary tumors were experimentally induced in female Wistar rats by repeated injections of estradiol dipropionate. The hypothalamus and pituitary tumors were studied simultaneously by fluorescence histochemistry and immunohistochemistry. The pituitary gland became larger with a concomitant increase of serum prolactin in proportion to the dose of estrogen. Estrogen-induced pituitary tumor exhibited a proliferating prolactin cells by the peroxidase immunohistochemical method. Ultramicroscopical findings showed that these tumor cells were in an extremely hyperfunctional state. The dopamine neuronal perikarya in the hypothalamic arcuate nucleus and their terminals in the external layer of the median eminence were examined by fluorescence histochemistry in the rats bearing estrogen induced pituitary tumor and it was concluded that in our experimental conditions, estrogen effected directly on pituitary rather than on the hypothalamus and consequently dopamine synthesis in the arcuate neurons and its release into portal capillaries were accerelated simultaneously in order to inhibit prolactin secretion from tumor cells.     

Pathogenesis of experimentally induced asbestos mesothelioma in rats

Fragments of parietal and visceral pleura were studied by total films preparation, light microscopy and SEM at different times after intrapleural injection of asbestos in Wistar rats. Pleural rat mesothelium in histological slices consists normally of one layer of oblong cells. By SEM the cells are flat and coated with microvilli of different lengths. In total films the parietal mesothelium was composed of large polygonal cells covering intercostal spaces and small cells covering spaces over the ribs. Inflammatory reaction and permanent pathological regenerative processes were observed in the mesothelium during 24 months after inoculation of asbestos fibres. Different lesions which we regarded as preneoplastic or premesotheliomatous were observed against the background of or without these processes. They were diffuse irregular hyperplasia and proliferation of epithelium-like or fibroblast-like cells and focal nodous proliferates composed of such cells with various morphological structures. The number of thymidine-labelled cells was significantly more inside the proliferates than in the surrounding tissue. They were confirmed by SEM and histological slices of the same fields. Chronic pathological regeneration of pleural mesothelium could be the background against which preneoplastic lesions and mesotheliomas develop easily.     

Induction of delayed hypersensitivity reactions in cancer patients by cholesterol-hemisuccinate-treated autologous tumor cells

Cholesterol-hemisuccinate (CHS) incorporated into tumor cells increases membrane lipid microviscosity and confers enhanced immunogenicity, which can be manifested by delayed hypersensitivity skin reactions. Skin testing was performed in 30 patients with various advanced malignant tumors. Patients were given intradermal injections of 10(6) autologous, irradiated, CHS-treated tumor cells. Control injections consisted of untreated irradiated tumor cells, CHS-treated autologous normal peripheral lymphocytes, or CHS-treated autologous normal tissues. For all patients tested, strongly positive skin reactions were observed when CHS-treated tumor cells were used. Untreated irradiated cells gave negative or very weakly positive reactions. In all cases, normal CHS-treated cells did not elicit any observable skin reactions. CHS-treated cells may have unmasked tumor-associated antigens to which patients may elicit immunologic responses.     

Regression of primary murine colon cancer and occult liver metastasis by intralesional injection of lyophilized preparation of insect cells producing murine interferon-beta

We investigated the efficacy of interferon (IFN)-beta therapy against colon cancer using a novel approach mediated by a lyophilized preparation of High 5 (H5) insect cells transduced with a recombinant baculovirus encoding murine IFN-beta (H5BVIFN-beta). The orthotopic model of CT-26 murine colon cancer in syngeneic BALB/c mice was used in the study, and H5BVIFN-beta was intratumorally delivered. Two injections of H5BVIFN-beta (on days 14 and 21 after tumor cell implantation), but not lyophilized H5 cells (control), significantly reduced the size of cecal tumors and the number of liver metastases. Immunohistochemical analysis revealed that cecal tumors injected with saline or H5 contained many proliferating cells (PCNA+) and few apoptotic cells (TUNEL+). In sharp contrast, H5BVIFN-beta-treated tumors contained fewer PCNA+ cells and significantly more TUNEL+ cells. The H5BVIFN-beta-treated tumors were infiltrated by a large number of CD8+ and F4/80+ cells and expressed a high level of inducible nitric oxide synthase (iNOS). Immunofluorescent double staining technique demonstrated a significant increase of apoptotic endothelial cells (CD-31+/TUNEL+) in tumors treated with H5BVIFN-beta. In conclusion, the data show that intralesional injections of H5BVIFN-beta can suppress the progressive growth of established orthotopic tumors of colon cancer cells and occult liver metastasis. The therapeutic effects directly correlate with destruction of tumor vasculature.     

Ultrastructural analysis of human plasmacytoma cells prepared on affinity beads after exposure to anti-idiotype antibodies

Mononuclear cells from a bone marrow infiltrated by plasmacytoma cells were examined by electron microscopy. An analysis was performed according to different cytoarchitectural forms of the endoplasmic reticulum (ER). Six types of plasma cells could be distinguished. The bone marrow cells were treated with an anti-idiotype antiserum from a guinea pig prepared against the patient's monoclonal serum protein and with a FITC conjugated anti-guinea pig antiserum from the rabbit as second layer. Then the cells were passed through an affinity gel column with anti-FITC antibodies. The original preparation and the cells separated on the affinity gel were analysed by electron microscopy. It was found that an ultrastructurally distinct type of plasma cell was enriched 3.5-fold over the original sample by the separation procedure.     

Delay of tumor growth in hamsters treated with lynestrenol and effect of Staphylococcus aureus Cowan A.

The action of lynestrenol, a synthetic progesterone-like substance, was studied in Syrian golden hamsters inoculated with cells transformed by herpes simplex virus type I. Daily ip injections of 1 mg lynesterol/kg delayed tumor growth. Lynestrenol alone only slightly increased the survival of the animals. This effect was more marked when the animals were also given injections of Staphylococcus aureus Cowan A. Survivors showed an increased resistance to challenge with infected cells. Hamsters previously given injections of killed transformed cells and then challenged with living cells showed a facilitation of tumor growth. This facilitation was inhibited by lynestrenol alone or combined with S. aureus Cowan A.     

The effects of protease I of Aspergillus oryzae (brinase) on membrane permeability and growth of Landschütz ascites tumour cells

Landschütz ascites tumour cells treated in vitro with Protease I showed no changes in intracellular K and Na levels when the cells were analysed directly at the end of the 60-min incubation period. If, however, the cells were washed twice in the saline medium before analysis significant losses of K, apparently in exchange for Na, were found to take place. Controls incubated in the absence of Protease I and similarly treated showed no such K loss. Viability tests, using lissamine green, indicated that the observed changes were not due to dead cells. These findings are interpreted as being due to an alteration in the plasma membrane associated with increased sensitivity to mechanical trauma, which in turn leads to secondary loss of K. Increases in UV absorbance in the 260 nm region were noted in the supernatant medium of treated cells. Normal mice weighing 30 g tolerated intraperitoneal injections of 0.3 mg Protease I and under, in repeated daily doses, but a single injection of 0.4 (i.e. 13 mg/kg) was lethal within 2 hours. The presence of tumour cells (7.5 mg dry wt) intraperitoneally was found to protect mice against repeated daily injections of this normally lethal dose, indicating that the enzyme is quickly adsorbed onto the tumour cells. No effect on tumour growth in vivo was found when single injections of Protease I were administered intraperitoneally at the same time as the tumour, but when additional doses of the enzyme were given on the two subsequent days, the 7-day yield of tumour cells was significantly increased.     

Analysis of natural killer cell cytotoxicity of cancer patients treated with recombinant interferon

Peripheral blood natural killer (NK) cell cytotoxicity of 24 cancer patients was studied prior to and after single and multiple injections of various doses of human leukocyte recombinant interferon-alpha clone A (IFN-alpha rA). The NK cell cytotoxicity of all cancer patients declined consistently 4 and 8 hours after a single injection of IFN-alpha rA. Twenty-four hours after the injection of IFN-alpha rA, NK cell cytotoxicity of patients with low NK cell phenotype (NK-LR) was significantly augmented, whereas that of patients with medium (NK-MR) or high (NK-HR) NK phenotype was depressed. After multiple injections of IFN-alpha rA, depression of NK cell cytotoxicity was observed in a number of NK-MR and NK-HR patients, but in some patients with NK-LR phenotype, further potentiation was observed. No direct correlation between the NK cell augmentation and serum IFN levels was detected. In in vitro studies, IFN-alpha rA, when added to cultures of target and effector cells of normal individuals in a dose of 10(3) U/ml, was efficient in augmenting NK cell cytotoxicity. NK cell cytotoxicity of cancer patients could also be augmented by the IFN-alpha rA preparation; however, this augmentation occurred only prior to in vivo IFN-alpha rA therapy. After IFN-alpha rA in vivo therapy, their NK cells became refractory to further in vitro IFN-alpha rA treatment.     

Reduction of hepatic metastases in rabbits by administration of an oily anticancer agent into the portal vein

We studied a prophylactic chemotherapy against hepatic metastases arising from the shedding of tumor cells into the portal circulation. The therapy was done with a lymphographic oily contrast medium, Lipiodol, and a high molecular weight anticancer agent named poly(styrene-maleic acid) copolymer conjugated neocarzinostatin (SMANCS), developed in our laboratory. SMANCS was dissolved in Lipiodol by sonication (SMANCS/Lipiodol, 1 mg of SMANCS in 1 ml of Lipiodol). Twelve rabbits were simply inoculated with the highly malignant carcinoma VX-2. Fifteen rabbits were given injections of SMANCS in glucose and Lipiodol into the portal vein and were subsequently inoculated with the tumor cells. Eighteen were given injections of SMANCS/Lipiodol and then the tumor cells. These rabbits were killed 12 days later. Thirteen were given injections of the tumor cells alone and were allowed to survive. Sixteen were given injections of SMANCS/Lipiodol and then with the tumor cells; they were allowed to survive. Rabbits given injections of SMANCS/Lipiodol before tumor inoculation had significantly fewer (P less than 0.001) metastases than those not treated or those given SMANCS in glucose and Lipiodol. Survival was significantly longer [P less than 0.005; 36.0 +/- 7.7 (SD) days] with SMANCS/Lipiodol before tumor inoculation than without treatment [23.5 +/- 3.0 days]. SMANCS/Lipiodol has a prolonged anticancer effect because it remains in the portal vein and allows sustained drug release from the oil (Lipiodol) to aqueous spaces. Hepatic metastases might be prevented by portal administration of the appropriate oily anticancer agent.     

C-type virus particles in urethan-induced pulmonary and renal carcinomas, in cell-graft-transmitted carcinosarcomas, and in filtrate-induced lymphomas in mice.

Repeated injections of urethan into suckling BALB/c mice induced multiple papillary adenocarcinomas in the lungs and kidneys. When the pulmonary tumors were transplanted i.p. by cell graft into 6 suckling BALB/c mice, they induced disseminated carcinosarcomas within the peritoneal cavity in all inoculated animals. Tumors resulting from the transplantation of tumor cells were used for preparation of filtered extracts. The filtrates were inoculated into 6 suckling BALB/c mice and induced generalized malignant lymphomas in all animals. The primary urethan-induced pulmonary and renal tumors, the carcinosarcomas that resulted from i.p. cell transfer, and also the generalized malignant lymphomas induced by inoculation of filtered extracts contained C-type virus particles. Theoretically, it could be assumed that both the primary urethan-induced pulmonary and renal tumors, as well as the cell-graft-induced peritoneal carcinosarcomas, contained the C-type virus particles as passengers, not necessarily related etiologically to the tumors in which they were found. It is quite likely, however, that these virus particles were etiologically related to the filtrate-induced malignant lymphomas in which they were also found.     

Susceptibility to and escape from complement-mediated lysis of guinea-pig hepatoma line-10.

Rabbit xenoantisera produced against diethyl-nitrosamine-induced strain-2 guinea-pig hepatoma line-10 cells (L-10) according to various immunization schedules were compared for their cytotoxicity on L-10 cells in the presence of guinea-pig complement. The highest activity was obtained with antisera (Cx-1) produced by repeated intravenous injections of living L-10 cells at high cell dosage, whereas intramuscular injections of living or glutaraldehyde-treated L-10 cells at similar frequency and cell dosage were less effective for the production of cytotoxic antibodies against L-10 cells. Intravenous injections of smaller cell doses were less effective. The cytotoxic antibody in Cx-1 antiserum was shown to be IgG by various methods including gel filtration on Sephadex G-200, ion exchange chromatography and immunoelectrophoresis of purified tumor-specific antibodies. It was concluded that L-10 cells can be lysed by guinea-pig complement and tumor-specific antibodies (IgG). Some antisera contained IgM antibodies which were not cytotoxic. A decrease in susceptibility of L-10 cells to complement-mediated lysis was observed when the cells were maintained in vivo for a long period (more than 20 passage generations). This was due to a lower density of tumor antigens on the cell surface. When tumor cells were treated with a second antibody directed against rabbit IgG or F(ab')2, cytotoxicity of Cx-1 antisera was completely abolished.     

Patterns of haemopoietic recovery after stress--II. Treatment with fluorouracil

The mouse haemopoietic system is not permanently damaged by repeated injections of cytotoxic fluorouracil. It contains approximately normal numbers of nucleated femoral and spleen colony-forming cells (CFUs) after seven monthly injections of the drug and normal numbers of high proliferation potential colony-forming cells (HPP-CFC) after five serial injections. Furthermore, the mouse is fully fertile after seven injections of fluorouracil. The mouse recovers quickly after treatment because it regenerates from cells which were not killed by the drug. Within 14 days of treatment with fluorouracil there are almost twice the normal number of femoral macrophage and high proliferation potential colony-forming cells (M-CFC and HPP-CFC). These numbers then fall but are returning to normal 6 weeks after the drug was administered. In this quick recovery the response of the haemopoietic system differs from its response to the loss of blood cells caused by sub-lethal irradiation, or lethal irradiation, or treatment with busulphan. When mice are treated twice with fluorouracil, the second injection 14 days after the first, the number of femoral M-CFC two days after the second injection, is 16-fold the number in controls, but the number of femoral HPP-CFC is only twice the number in controls. When the interval between the two injections is 21 days, the number of femoral M-CFC is almost 8% of that of mice treated once, but the number of HPP-CFC is 67%. The characteristic response of each type of cell to repeated treatment with fluorouracil is probably due to the number of its precursors which are killed by the drug and to the interval between successive injections. A second injection of fluorouracil, 28 days after the first, speeds the growth rate of HPP-CFC. Their doubling time is 6 h shorter than that of mice treated once. Haemopoietic tissue from mice treated repeatedly with fluorouracil can only outgrow normal marrow under certain conditions. The nature of these conditions and the mechanisms involved are discussed in the light of contradictory findings.