A lesson on induction of hypothermia and measurement of efficacy

Brain injuries caused by stroke are common and costly in human and resource terms. The result of stroke is a cascade of molecular and physiological derangement, cell death, damage and inflammation in the brain. This, together with infection, if present, commonly results in patients having an increased temperature, which is associated with worse outcome. The usual clinical goal in stroke is therefore to reduce temperature to normal, or below normal (hypothermia) to reduce swelling if brain pressure is increased. However, research evidence does not yet conclusively show whether or not cooling patients after stroke improves their longer-term outcome (reduces death and disability). It is possible that complications of cooling outweigh the benefits. Cooling therapy may reduce damage and potentially improve outcome, and head cooling targets the site of injury and may have fewer side effects than systemic cooling, but the evidence base is unclear.The recent study by Poli and colleagues [1] is part of a suite of iCOOL studies in ischaemic and haemorrhagic stroke, conducted at Heidelberg and linked to EuroHYP-1 (European multicentre, randomised, phase III clinical trial of therapeutic hypothermia plus best medical treatment versus best medical treatment alone for acute ischaemic stroke [2]). Altogether the studies tested four different methods of inducing hypothermia for speed of brain cooling, feasibility and safety. Rhinochill and the Sovika head and neck cooling device [1] and cold infusions compared with Rhinochill (Rhinochill, Wallisellen, Switzerland, EU) (iCOOL 1 {"type":"clinical-trial","attrs":{"text":"NCT01573117","term_id":"NCT01573117"}}NCT01573117 [1]), EMCOOLS Flex.Pads (EMCOOLS, Brucknerstrasse 6/7a, 1040 Vienna, Austria) (iCOOL 2 {"type":"clinical-trial","attrs":{"text":"NCT01584167","term_id":"NCT01584167"}}NCT01584167) and EMCOOLS Brain.Pad (EMCOOLS, Brucknerstrasse 6/7a, 1040 Vienna, Austria) (iCOOL 3 {"type":"clinical-trial","attrs":{"text":"NCT01584180","term_id":"NCT01584180"}}NCT01584180). The methods of inducing hypothermia that are now included in the protocol for EuroHYP-1 are cold infusion (20 ml/kg 4°C isotonic sodium chloride or Ringer’s lactate over 30 to 60 minutes) with optional use of EMCOOLs Brain.Pad [3].There has been an ongoing quest for methods of therapeutic cooling that reduce temperature rapidly, are portable and easily instigated and/or have the least side effects. Cold infusion as a method of inducing cooling has most often been studied in cardiac arrest but is currently being used in clinical trials of therapeutic hypothermia in stroke (EuroHYP-1 [3]) and traumatic brain injury (Eurotherm3235Trial [4] and POLAR ({"type":"clinical-trial","attrs":{"text":"NCT00987688","term_id":"NCT00987688"}}NCT00987688)). The attraction is that it is a relatively low-tech, readily available, portable method, requiring only a means of keeping the infusion fluid at 4°C. Direct brain cooling has a long history but there are few randomised controlled trials and most are of low quality [5]. One of the attractions has been the assumption that direct brain cooling has fewer side effects than systemic cooling but this has not been established [5]. Various methods of nasopharyngeal cooling have been reported in the literature [5], including Rhinochill and nasal [6] and pharyngeal balloons [7]. Rhinochill has been studied most (for example, [8]) and there is very limited human data on the pharyngeal balloon device [7] or the pharyngeal cooling system of Takeda. Springborg and colleagues [6] report the use of QuickCool nasal balloons in a mixed group of hyperthermic brain-injured patients. Temperature was measured in the oesophagus, bladder and (in some patients) intracranially. The goal of normothermia was not reliably achieved. As with Poli and colleagues [1], this research raises questions about bladder temperature as a proxy for intracranial temperature.Perfluorocarbons are costly and their use in Rhinochill has been questioned on environmental grounds [9]. Although in the overall context of medical interventions Rhinochill may not have major environmental impact, this nevertheless warrants consideration. Use of Rhinochill requires patients to be intubated and is contraindicated with base of skull fracture, which limits its use in traumatic brain injury.Recently another method of nasopharyngeal cooling has been reported by Fontaine and colleagues [10], with experimental evidence and a human case report. This method uses adiabatic expansion of gas pressure; 1 L compressed carbon dioxide delivered via a nasal cannula. Temperature is reduced because as the gas expands the pressure reduction transfers energy as work (very rapidly) and not as heat, although in practice there is some heat transfer as insulation is not perfect. Carbon dioxide is of course also a greenhouse gas and using it in this way requires patients to be intubated. Compressed air and oxygen were tested experimentally as alternatives and found to remove considerably less heat than carbon dioxide, but in vivo temperature differences were not significant. In their study, Poli and colleagues [1] show very nicely how different sites of temperature measurement reflect temperature change differently using intravenous and nasopharyngeal cooling. Where intracranial temperature is the key temperature of interest, as it arguably is in stroke and traumatic brain injury, their data show the importance of measuring this. As yet, however, there does not seem to be much appetite for targeting therapeutic cooling to intracranial temperature - invasive measurement is not clinically warranted in less severe stroke and traumatic brain injury - and core body temperature is the usual feedback parameter. In this case Poli and colleagues’ data [1] strongly suggest oesophageal temperature is the best proxy for intracranial temperature. One difficulty with this site of measurement is that with longer-term cooling, if drugs are given nasogastrically, this will affect the temperature readings [11]. Another is having to site both a nasogastric tube and an oesophageal temperature probe with attendant increased risk of sinusitis and abrasions. There have been moves to produce a nasogastric tube suitable for feeding/drug instillation, aspiration and temperature measurement but to our knowledge such a potentially useful device is not yet in commercial production.The authors are to be congratulated on their comprehensive measurement of temperature reduction efficacy and clear presentation of data measured at multiple sites. The challenge is to move from evidence of efficacy (temperature reduction) to evidence of effectiveness and improved patient outcomes.     

Autocrine secretion of GM-CSF in acute myeloblastic leukemia

Three cases of acute myeloblastic leukemia (AML) were identified in which clonogenic cells proliferated autonomously in vitro. Cells from two of these cases were found to secrete a colony-stimulating factor (CSF) that was immunologically and molecularly related to GM-CSF. Growth of AML-CFU could be blocked by the addition of a neutralizing antiserum to GM-CSF. Northern blot hybridization of leukemic cell mRNA with a cDNA probe for the GM-CSF gene revealed a 1-kb message identical in size to the normal GM-CSF message in stimulated T cells. No GM-CSF message was detected in the third case. These results indicate that constitutive expression of the GM-CSF gene, apparently by leukemic cells, can result in autonomous in vitro proliferation of AML-CFU in some cases of AML.     

Clonal, nonconstitutional rearrangements of the MLL gene in infant twins with acute lymphoblastic leukemia: in utero chromosome rearrangement of 11q23

Rearrangements of chromosome band 11q23 are common in infant leukemias, comprising more than 70% of the observed chromosome abnormalities in children less than 1 year of age. The MLL gene, which is located at the 11q23 breakpoint in infant, childhood, and adult acute leukemias, has been cloned and has homology to the Drosophila trithorax gene. The breakpoints in MLL are restricted to an 8.3-kilobase pair (kb) region of the gene that is involved in translocations with as many as 29 other chromosomal regions in a number of phenotypically distinct acute leukemias. We have detected an identical, clonal, nonconstitutional rearrangement of the MLL gene in peripheral blood cells from a pair of female infants twins with acute lymphoblastic leukemia (ALL) and a t(11;19)(q23;p13.3). The detection of nonidentical IGH rearrangements suggests that the MLL rearrangement took place in a B-cell precursor or hematopoietic stem cell in one twin which was transferred in utero to the other fetus resulting in ALL with an identical aneuploid karyotype in both infants. We speculate that the other MLL-related infant leukemias may also develop in utero, and that the rearrangements may occur consistently in stem cells or early precursor cells, accounting for the frequency of mixed-lineage leukemia in infants.     

Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. I. Stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF-beta

Long-term marrow cultures (LTMC) allow the proliferation and differentiation of primitive human hematopoietic progenitor cells to be maintained for many weeks in the absence of exogenously provided hematopoietic growth factors. Previous investigations focused on defining various types of cells that are present in this culture system and on measuring the cycling behavior of the different subpopulations of colony-forming cells maintained within it. These studies suggested that mesenchymal stromal elements derived from the input marrow play a key role in regulating the turnover of the most primitive, high- proliferative potential erythroid and granulopoietic colony-forming cells that are found almost exclusively in the adherent layer of LTMC. In this study we show that the re-entry into S-phase of these primitive hematopoietic progenitors that occurs after each weekly medium change is due to an as yet undefined constituent of horse serum, which is absent from fetal calf serum. However, this effect is not unique to the factor present in horse serum. It is also elicited by the addition to LTMC of several well-defined growth regulatory molecules, ie, platelet- derived growth factor (PDGF), interleukin-1 (IL-1), transforming growth factor alpha (TGF-alpha), and IL-2. None of these was able to stimulate hematopoietic colony-forming cells in methylcellulose assays, although all have known actions on mesenchymal cells including, in some cases, the ability to increase production of growth factors that can stimulate primitive high-proliferative potential hematopoietic progenitors in clonogenic assays. Interestingly, a stimulating effect was not obtained after addition of endotoxin to LTMC. TGF-beta, a direct-acting negative regulator that acts selectively on primitive hematopoietic progenitor cells if added to LTMC simultaneously with new medium or IL-1, blocked their stimulating activity. These results suggest a model in which indirect, local modulation of both positive and negative regulatory factors via effects on mesenchymal elements determines the rate of turnover of adjacent populations of very primitive hematopoietic cells that are normally maintained in a quiescent state in vivo.     

Indication of an involvement of interleukin-1 beta converting enzyme- like protease in intramedullary apoptotic cell death in the bone marrow of patients with myelodysplastic syndromes

Our previous studies using in situ end labeling (ISEL) of fragmented DNA revealed extensive apoptotic cell death in the bone marrows (BM) of patients with myelodysplastic syndromes (MDS) involving both stromal and hematopoietic cells. In the present report we show greater synthesis of interleukin-1 beta (IL-1 beta) in 4 hour cultures of density separated BM aspirate mononuclear (BMAM) cells from MDS patients as compared to the cultures of normal BM from healthy donors or lymphoma patients (1.7 +/- 0.37 pg/10(5) cells, n = 29 v 0.42 +/- 0.24 pg/10(5) cells, n = 11, respectively, P = .049). Further, these amounts of IL-1 beta in MDS showed a significant correlation with the extent of apoptosis detected by ISEL in corresponding plastic embedded BM biopsies (r = .480, n = 30, P = .007). In contrast normal BMs did not show any correlation between the two (r = .091, n = 12, P = .779). No significant correlation was found between the amounts of IL-1 beta and % S-phase cells (labeling index; LI%) in MDS determined in BM biopsies using immunohistochemistry following in vivo infusions of iodo- and/or bromodeoxyuridine. Neither anti-IL-1 beta antibody nor IL-1 receptor antagonist blocked the apoptotic death of BMAM cells in 4 hour cultures (n = 5) determined by ISEL (apoptotic index; AI%), although the latter led to a dose-dependent accumulation of active IL-1 beta in the culture supernatants. On the other hand, a specific tetrapetide- aldehyde inhibitor of ICE significantly retarded the apoptotic death of BMAM cells at 1 mumol/L in 5/6 MDS cases studied (AI% = 2.99 +/- 0.30 in controls v 1.58 +/- 0.40 with ICE-inhibitor, P = .05) and also reduced the levels of active IL-1 beta synthesized (5.59 +/- 2.63 v 2.24 +/- 0.93 pg/10(6) cells, respectively). In normal cells, neither IL-1 blockers nor the ICE inhibitor showed any effect on the marginal increase in apoptosis observed in 4 hour cultures. Our data thus suggest a possible involvement of an ICE-like protease in the intramedullary apoptotic cell death in the BMs of MDS patients.     

Immunomorphologic analysis of bone marrow biopsies after treatment with 2-chlorodeoxyadenosine for hairy cell leukemia

Treatment of hairy cell leukemia with 2-chlorodeoxyadenosine (2-CdA) induces complete remissions in 85% of patients. Complete remission has been defined as the absence of hairy cells in the bone marrow after routine morphologic examination. To determine if hairy cells could be detected in complete remission bone marrows using immunohistochemical techniques with antibodies L26 (CD20) and DBA.44, 154 bone marrow biopsies performed between 3 months and 25 months after therapy were studied. Of the biopsies, 50% exhibited staining with L26 and/or DBA.44 in five or more cells with morphologic features of hairy cells. Minimal residual disease was usually less than 1% of the total cellular population. DBA.44-positive cells were demonstrated in 91% of the biopsies, although in 48% of these the morphologic features of the positive cells were not sufficiently distinctive for hairy cells. The proportion of biopsies with residual hairy cells was similar over the 25 months of follow up, indicating a relatively stable amount of residual disease. Immunomorphologic analysis is a more sensitive method for detecting residual hairy cells than morphology alone. Although further follow up is necessary to determine the clinical significance of the L26/DBA.44-positive staining in cells with and without distinctive morphologic features of hairy cells, we conclude that many patients in a stable clinical remission may have residual hairy cells.