'Iron-saturated' lactoferrin is a potent natural adjuvant for augmenting cancer chemotherapy

Bovine lactoferrin (bLf), an iron-containing natural defence protein found in bodily secretions, has been reported to inhibit carcinogenesis and the growth of tumours. Here, we investigated whether natural bLf and iron-saturated forms of bLf differ in their ability to augment cancer chemotherapy. bLf was supplemented into the diet of C57BL/6 mice that were subsequently challenged subcutaneously with tumour cells, and treated by chemotherapy. Chemotherapy eradicated large (0.6 cm diameter) EL-4 lymphomas in mice that had been fed iron-saturated bLf (here designated Lf(+)) for 6 weeks prior to chemotherapy, but surprisingly not in mice that were fed lesser iron-saturated forms of bLf, including apo-bLf (4% iron saturated), natural bLf (approximately 15% iron saturated) and 50% iron-saturated bLf. Lf(+)-fed mice bearing either EL-4, Lewis lung carcinoma or B16 melanoma tumours completely rejected their tumours within 3 weeks following a single injection of either paclitaxel, doxorubicin, epirubicin or fluorouracil, whereas mice fed the control diet were resistant to chemotherapy. Lf(+) had to be fed to mice for more than 2 weeks prior to chemotherapy to be wholly effective in eradicating tumours from all mice, suggesting that it acts as a competence factor. It significantly reduced tumour vascularity and blood flow, and increased antitumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf(+) bound to the intestinal epithelium and was preferentially taken up within Peyer's patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-gamma, as well as nitric oxide that have been reported to sensitize tumours to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron to be effective.     

Development of a pharmaceutical apotransferrin product for iron binding therapy.

High-dose chemotherapy of patients with haematological malignancies results in extracellular iron accumulation and appearance of non-transferrin-bound iron, which is thought to predispose the patients to septic infections and contribute to organ toxicity. We describe the development of a human plasma-derived apotransferrin product for iron binding therapy. The product is purified from Cohn fraction IV of human plasma by two ion exchange chromatography steps and ultrafiltration. The process comprises solvent detergent treatment as the main virus inactivation step and 15 nm virus filtration and polyethylene glycol precipitation as removal steps for physico-chemically resistant infectious agents. Product characterization by electrospray and MALDI-TOF mass spectrometry indicated no other chemical modifications than N-linked glycan chains and disulphide bonds, except minor oxidation. The purity of the product was more than 98%, main impurities being IgG, IgA and hemopexin. The product had intact iron binding capacity and native conformation. A stable liquid formulation for the finished product was developed. The product has proved safe and well tolerated in early clinical trials in iron binding therapy.     

Organ donation from intensive care units in England.

OBJECTIVE--To audit all deaths in intensive care units (excepting coronary care only and neonatal intensive care units) in England to assess potential for organ procurement. DESIGN--An audit in which 14 regional health authorities and London special health authorities each designated a regional liaison officer to identify intensive care units and liaise with Department of Health and the Medical Research Council''s biostatistics unit in distribution, return, and checking of audit forms. Audit took place from 1 January to 31 March 1989 and will continue to 31 December 1990. SETTING--278 Intensive care units in England. PARTICIPANTS--Colleagues in intensive care units (doctors, nurses, coordinators, and others), who completed serially numbered audit forms for all patients who died in intensive care. RESULTS--The estimated number of deaths in intensive care units was 3085, and validated audit forms were received for 2853 deaths (92%). Brain stem death was a possible diagnosis in only 407 (14%) patients (about 1700 cases a year) and was confirmed in 282 (10%) patients (an estimated 1200 cases a year). Half the patients (95% confidence interval 45% to 57%) in whom brain stem death was confirmed became actual donors of solid organs. Tests for brain stem death were not performed in 106 (26%) of 407 patients with brain stem death as a possible diagnosis, and general medical contraindication to organ donation was recorded for 48 (17%) of 282 patients who fulfilled brain stem death criteria before cessation of heart beat. The criteria were fulfilled before cessation of heart beat and in the absence of any general medical contraindication to organ donation in 234 patients, 8% of those dying in intensive care (an estimated 1000 cases a year). Consent for organ donation was given in 152 (70%) of 218 cases (64% to 76%) when the possibility of organ donation was suggested to relatives. In only 14 out of 232 families (6%; 3% to 9%) was there no discussion of organ donation with relatives. Corneal suitability was recorded as "not known" in a high proportion (1271; 45%) of all deaths and intensive care units reported only 123 corneal donors (4% of all audited deaths). CONCLUSION--When brain stem death is a possible diagnosis tests should always be carried out for confirmation. Early referral to the transplant team or coordinator should occur in all cases of brain stem death to check contraindications to organ donation. There should be increased use of asystolic kidney donation, and patients should be routinely assessed for suitability for corneal donation. Finally, more publicity and education are necessary to promote consent.     

Prognosis of patients receiving intensive care for lifethreatening medical complications of haematological malignancy

The mortality of patients admitted to intensive care units with haematological malignancy is high. A humane approach to the management of the critically ill as well as efficient use of limited resources requires careful selection of those patients who are most likely to benefit from intensive care. To delineate more accurately the factors influencing outcome in these patients the records of 60 consecutive admissions to the intensive care unit (37 male, 23 female) with haematological malignancy were reviewed retrospectively. Fifty patients were in acute respiratory failure, most commonly (34 patients) with a combination of pneumonia and septicaemic shock. The severity of the acute illness was assessed by the APACHE II (acute physiology and chronic health evaluation II) score and number of organ systems affected. Thirteen patients survived to leave hospital. The mortality of patients with haematological malignancy was consistently higher than predicted from a large validation study of APACHE II in a mixed population of critically ill patients. Moreover, no patient with an APACHE II score of greater than 26 survived. Mortality among the 22 patients with relapsed malignancy (21 deaths), was significantly higher than among the 35 patients at first presentation (26 deaths). On discharge from the intensive care unit all survivors had responded well to chemotherapy and had normal or raised peripheral white cell counts. They included seven patients who had recovered from leucopenia (white cell count <0.5 × 10⁹/1). In contrast, 36 of the 47 patients who died were leucopenic at the time of death.The overall mortality of critically ill patients with haematological malignancy is higher than equivalently ill patients without cancer. The dysfunction of an increasing number of organ systems, an APACHE II score of greater than 30, failure of the malignancy to respond to chemotherapy, and persistent leucopenia all point to a poor outcome.     

Measurement by HPLC of Desferrioxamine-Available Iron in Rabbit Kidneys to Assess the Effect of Ischaemia on the Distribution of Iron Within the Total Pool

A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1-10µMolar region could be accurately and reproducibly measured using this technique.

The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen.     

Measurement by HPLC of Desferrioxamine-Available Iron in Rabbit Kidneys to Assess the Effect of Ischaemia on the Distribution of Iron Within the Total Pool

A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1‐10µMolar region could be accurately and reproducibly measured using this technique.

The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen.     

Does HIV status influence the outcome of patients admitted to a surgical intensive care unit? A prospective double blind study.

OBJECTIVES: (a) To assess the impact of HIV status (HIV negative, HIV positive, AIDS) on the outcome of patients admitted to intensive care units for diseases unrelated to HIV; (b) to decide whether a positive test result for HIV should be a criterion for excluding patients from intensive care for diseases unrelated to HIV. DESIGN: A prospective double blind study of all admissions over six months. HIV status was determined in all patients by enzyme linked immunosorbent assay (ELISA), immunofluorescence assay, western blotting, and flow cytometry. The ethics committee considered the clinical implications of the study important enough to waive patients'' right to informed consent. Staff and patients were blinded to HIV results. On discharge patients could be advised of their HIV status if they wished. SETTING: A 16 bed surgical intensive care unit. SUBJECTS: All 267 men and 135 women admitted to the unit during the study period. INTERVENTIONS: None. MAIN OUTCOME MEASURES: APACHE II score (acute physiological, age, and chronic health evaluation), organ failure, septic shock, durations of intensive care unit and hospital stay, and intensive care unit and hospital mortality. RESULTS: No patient had AIDS. 52 patients were tested positive for HIV and 350 patients were tested negative. The two groups were similar in sex distribution but differed significantly in age, incidence of organ failure (37 (71%) v 171 (49%) patients), and incidence of septic shock (20 (38%) v 54 (15%)). After adjustment for age there were no differences in intensive care unit or hospital mortality or in the durations of stay in the intensive care unit or hospital. CONCLUSIONS: Morbidity was higher in HIV positive patients but there was no difference in mortality. In this patient population a positive HIV test result should not be a criterion for excluding a patient from intensive care.     

Treatment strategies in acute myeloid leukemia (AML). A. First-line chemotherapy

Chemotherapy remains the backbone of treatment in AML. Of all adult patients 65% go into remission and have a chance of longer survival and of even being cured. Among the responders, 25% can be cured by intensive and long-term chemotherapy. Since the effect of chemotherapy appears not to be limited to a special period of time, its antileukemic and curative potential could be further exploited by multiple-step chemotherapy combining double induction, intensified consolidation and long-term maintenance. Improved antimicrobial treatment and the use of hematopoietic growth factors may help making multiple-step chemotherapy practicable, thereby increasing the cure rate in AML.     

Iron overload of the liver by trimethylhexanoylferrocene in rats.

Iron-deficient female Wistar rats were fed a diet, which contained 0.5% trimethylhexanoylferrocene, over a 56-week period. This dietary iron loading resulted in a progressive siderosis and enlargement of the liver with a maximum iron content of 947.0 +/- 148.0 mg (vs. 0.07 +/- 0.04 mg in iron deficiency) and a maximum organ weight of 39.4 +/- 6.6 g (vs. 6.9 +/- 1.4 g in iron-deficient control rats). Up to 43 weeks, whole liver iron rose by increase in iron concentration (max. 28.0 +/- 6.1 mg/g wet weight, w.w.) as well as by enlargement of the organ. Afterwards whole liver iron increased solely by ongoing hepatomegaly. At the commencement of iron loading, stainable iron was almost exclusively stored by hepatocytes equally throughout all areas of the liver lobule. Later, the distribution of iron-loaded hepatocytes became strikingly periportal, and, in addition, Kupffer cells as well as sinus-lining endothelia began to store iron. Animals with a liver iron concentration of more than 10.4 +/- 0.75 mg/g w.w. showed no further increase in ferritin and haemosiderin within hepatocytes. Iron-burdened Kupffer cells/macrophages, however, accumulated permanently, hereby forming intrasinusoidal and portal siderotic nodules and areas. First signs of liver damage such as necrosis of single hepatocytes and mild fibrosis began at a liver iron concentration of 14.7 +/- 1.4 mg/g w.w. With advancement of iron loading, focal necrosis of hepatocytes and iron-burdened macrophages took place, and significant perisinusoidal as well as portal fibrosis developed. Cirrhosis, however, the final stage of impairment in iron overload of the liver in humans, could not be induced in this animal model up to now.     

Cryopreserved autologous bone marrow transplantation in patients with acute nonlymphoid leukemia: chemotherapy before harvesting is the main factor in delaying hematological recovery

We analyzed the kinetics of hematological recovery after autologous bone marrow transplantation in 13 patients with acute nonlymphoid leukemias (ANLL). A comparison was made with 31 patients with non-Hodgkin's lymphoma (NHL) whose bone marrow was harvested and cryopreserved, either at diagnosis or after intensive chemotherapy. Hematological recovery of ANLL patients was similar to that of pretreated NHL patients and significantly slower than that of untreated NHL patients. We suggest that chemotherapy before harvest (more than the possible decreased stem cell marrow potentiality resulting from the underlying disease) appears to be the main factor responsible for delayed recovery after autologous bone marrow transplantation in ANLL.     

Distribution and metabolism of iron in muscles of iron-deficient rats

Iron-deficiency anemia leads directly to both reduced hemoglobin levels and work performance in humans and experimental animals. In an attempt to observe a direct link between work performance and insufficient iron at the cellular level, we produced severe iron deficiency in female weanling Sprague-Dawley rats following five weeks on a low-iron diet. Deficient rats were compared with normal animals to observe major changes in hematological parameters, body weight, and growth of certain organs and tissues. The overall growth of iron-deficient animals was approximately 50% of normal. The ratio of organ weight: body weight increased in heart, liver, spleen, kidney, brain, and soleus muscle in response to iron deficiency. Further, mitochondria from heart and red muscle retained their iron more effectively under the stress of iron deficiency than mitochondria from liver and spleen. Metabolism of iron in normal and depleted tissue was measured using tracer amounts of⁵⁹Fe administered orally. As expected, there was greater uptake of tracer iron by iron-deficient animals. The major organ of iron accumulation was the spleen, but significant amounts of isotope were also localized in heart and brain. In all muscle tissue examined the⁵⁹Fe preferentially entered the mitochondria. Enhanced mitochondrial uptake of iron prior to any detectable change in the hemoglobin level in experimental animals may be indicative of nonhemoglobin related biochemical changes and/or decrements in work capacity.     

Lactobionic acid as an iron chelator: a rationale for its effectiveness as an organ preservant

Lactobionic acid, a major constituent of a solution used to preserve organs prior to transplantation, can chelate ferric iron. This is evident by its ability to solubilize iron as well as changes that occur in the UV-VIS spectra of iron in its presence. Relative to iron (III) chelated to EDTA, the lactobionic acid-iron (III) complex is less able to participate in the Fenton reaction as measured by formaldehyde generation from DMSO and bleaching of p-N,N-dimethylnitrosoaniline. Similar effects are seen with citrate and ATP, two substances which also appear to be able to ameliorate ischemia/reperfusion injury. These findings present a rationale for the effectiveness of lactobionic acid as an organ preservant.     

Ferrous iron is found in mesenteric lymph bound to TIMP-2 following hemorrhage/resuscitation

Extracellular iron has been implicated in the pathogenesis of post-injury organ failure. However, the source(s) and biochemical species of this iron have not been identified. Based upon evidence that distant organ injury results from an increase in intestinal permeability, we looked for ferrous iron in mesenteric lymph in anesthetized rats undergoing hemorrhage and fluid resuscitation (H/R). Ferrous iron increased in lymph from 4.7 nmol/mg of protein prior to hemorrhage to 86.6 nmol/mg during resuscitation. Utilizing immuno-spin trapping in protein fractions that were rich in iron, we tentatively indentified protein carrier(s) of ferrous iron by MALDI-TOF MS. One of the identified proteins was the metalloproteinase (MMP) inhibitor, TIMP-2. Antibody to TIMP-2 immunoprecipitated 74% of the ferrozine detectable iron in its protein fraction. TIMP-2 binds iron in vitro at pH 6.3, which is typical of conditions in the mesentery during hemorrhage, but it retains the ability to inhibit the metalloproteases MMP-2 and MMP-9. In summary, there is a large increase in extracellular ferrous iron in the gut in H/R demonstrating dysregulation of iron homeostasis. We have identified, for the first time, the binding of extracellular iron to TIMP-2.     

Atrial fibrillation in β-thalassemia patients with a focus on the role of iron-overload and oxidative stress: A review

Cardiac complications including arrhythmia and especially atrial fibrillation (AF) are common causes of death in β-thalassemia patients. The main factor in the etiopathogenesis of these complications is iron overload, which results in increased oxidative stress. Although there is a known association between cardiac complications and iron overload in β-thalassemia patients, there is no comprehensive review on AF and excessive iron with a focus on oxidative stress in these patients. The aim of this article was to review the different aspects of AF in β-thalassemia patients with a focus on the prevention and treatment of AF by using iron chelators and/or anti-oxidants. AF in β-thalassemia patients is more common than in the general population. One of the most important causes of AF is cardiac iron overload and the harmful effects of increased oxidative stress. Iron-induced AF can be reversed by using an intensive iron chelation regimen. Based on a few experimental studies, the combination of iron chelators with some anti-oxidants, including NAC, vitamin C, and acetaminophen, can lead to improved cardiac protection. However, the effect of such combinations on cardiac arrhythmias should be further evaluated with animal and human studies.     

Retrospective evaluation of the incidence and severity of hemosiderosis in a large captive lemur population

Significant concern has been generated about the susceptibility of captive lemurs to iron storage disease, which has led some researchers to propose husbandry changes regarding dietary iron. In the current study we sought to determine the history, severity, and prevalence of iron storage disease within a large captive lemur population. Iron concentration and hemosiderin accumulation in a target organ, the liver, were assessed in necropsy specimens from 15 different species (n=153) of lemurs over a 12-yr period at the Duke University Primate Center. Banked liver tissue was used to quantify liver iron concentration (LIC) via neutron activation analysis (NAA). Prussian blue staining was used to accentuate the presence of liver iron for evaluation using an established scoring system. Of the 153 reports examined, 49 (32%) of the animals were considered positive for the presence of hemosiderin in the liver, lymph node, duodenum, and kidney, with 36 of the 49 (73%) showing deposition of iron in the liver. Total iron scores (TIS) ranged from 0.3+/-0.3 in Lemur catta to 33.3+/-1.7 in Cheirogaleus medius. The mean LIC ranged from 209+/-1.4 microg/g wet weight in L. catta to 2957+/-414 microg/g in C. medius. Management practices may have contributed to some of the results observed in this study. Although evidence of excess iron deposition in the liver was present across several species studied, the levels were not as pervasive as previously reported in other captive lemur populations. Hemochromatosis was not observed, and excess iron was not related to the cause of death in any of the animals studied. The current findings suggest that iron overload in lemurs may be more complex than was previously believed.     

Extrakorporale Membranoxygenierung in der Intensivmedizin

Extracorporeal membrane oxygenation (ECMO) represents a valuable tool in the armamentarium of life and organ support measures in intensive care medicine. ECMO is used in fulminant pulmonary failure to prevent acute hypoxia and to allow CO₂ removal (veno-venous ECMO) or, in highly selected cases of acute cardiovascular failure, to maintain organ perfusion and gas exchange and to prevent imminent death (veno-arterial ECMO). The purpose of ECMO is to allow time for intrinsic recovery of the lungs and heart. Alternatively, ECMO can be used as bridging device until a suitable organ for transplantation is available or the implantation of an artificial heart is feasible. ECMO carries a high complication rate and its use should be limited to highly specialized centers. In rare cases, mobile ECMO systems can be used by dedicated teams which allow transportation of critically ill patients over longer distances to specialized institutions. There is no unequivocal evidence from controlled studies that the use of ECMO in adults improves survival in larger cohorts, but its use in selected cases can be life saving. Long-term survivors of ECMO therapy report significant impairments in health-related quality of life (HRQL) when compared to healthy controls. There is, however, a gradual recovery and improvement in HRQL over a prolonged period after discharge from the intensive care unit and severe limitations in cognitive or physical function are rare. The incidence of chronic post-traumatic stress disorder (PTSD) in patients after ECMO can reach up to 30%. PTSD has a major negative impact on HRQL outcomes of ECMO therapy. PTSD or other stress-related disorders need to be diagnosed and adequately treated to allow adequate recovery from the extreme illness these patients have survived.     

Multiple isoferritins in mouse liver: demonstration by polyacrylamide gel electrophoresis

Unfractionated pure ferritin isolated from the livers of female or male mice forms five narrow protein-positive bands on standard 5% polyacrylamide gel disc electrophoresis (pH 9.0). Since all of these sub-bands also contain iron, they are interpreted as being isoferritins. The multiple sub-bands are very unlikely to be artifactually generated by the analytical procedure used since they are not found in horse spleen ferritin when this is coelectrophoresed with female mouse liver ferritin. The present results provide an independnet indication that many isoferritins indeed can be found within a single organ.