Effect of mass of 111In-benzyl-EDTA monoclonal antibody on hepatic uptake and processing in mice.

In patients, the pharmacokinetic behavior of murine monoclonal antibodies has been observed to vary with the amount of antibody administered. It has been suggested that this reflects human recognition of the foreign mouse protein. We have found that the amount of antibody administered also influenced pharmacokinetic behavior when murine monoclonal antibody was administered to mice. p-Isothiocyanatobenzyl-EDTA, a new chelator which forms complexes with 111In that are stable in vivo, was conjugated to Lym-1, a murine anti-Burkitt's lymphoma monoclonal antibody. The pharmacokinetics of two doses (20 and 0.2 micrograms) of the 111In labeled radiopharmaceutical were studied in non-tumor bearing BALB/c mice. About 20% (0.04 microgram) of the 0.2-microgram dose, compared with 8% (1.6 micrograms) of the 20-micrograms dose, was found in the liver at 48 h after injection. Both doses demonstrated a biological half-life of approximately 120 h. At least 75% of the 111In was excreted by the kidneys, and essentially all 111In in the urine remained chelated by the EDTA portion of p-isothiocyanatobenzyl-EDTA. From these observations of a dose dependent uptake of this radiopharmaceutical by the liver we conclude that there is a recognition phenomenon in mice for this murine monoclonal antibody.

Development of humoral immune responses against a macrocyclic chelating agent (DOTA) in cancer patients receiving radioimmunoconjugates for imaging and therapy.

The development of stable immunoconjugates by the advent of macrocyclic metal chelating agents (DOTA) has enabled us to study the ability of 111In-DOTA-labeled monoclonal antibodies to detect tumor lesions in a pilot radioimmunolocalization study, as well as to evaluate the kinetics, toxicity, and efficacy of i.p. administered 90Y-DOTA-labeled murine monoclonal antibody in a Phase I/II clinical trial of advanced ovarian cancer. The development of serum sickness-like reactions in three of six treated patients, in the absence of previous monoclonal antibody administration, led us to study the potential immunogenicity of the new chelate. Six patients with ovarian cancer received 25 mg of HMFG1 monoclonal antibody coupled with 90Y-DOTA (doses of radioactivity, 15 to 25 mCi), administered i.p. Eight patients with various malignant tumors received low doses (220 micrograms to 1 mg) of monoclonal antibodies, labeled with 111In-DOTA, i.v. for imaging studies. Using a solid-phase enzyme-linked immunosorbent assay method, the immunogenicity of DOTA was evaluated. Serial dilutions of patients' sera, before and after imaging or therapy with DOTA-coupled monoclonal antibodies, as well as sera from patients who did not receive DOTA-coupled antibody, were screened on enzyme-linked immunosorbent assay plates coated with human serum albumin (HSA), HSA-2-iminothiolane, and HSA-2-iminothiolane-benzyl-DOTA. All patients treated with i.p. monoclonal antibody developed anti-DOTA antibodies. Four of eight patients who received i.v. "imaging" doses of DOTA-coupled monoclonal antibody developed antibodies against DOTA. The levels of anti-DOTA response correlated with the amount of injected radioimmunoconjugate (r = 0.889, P less than 0.001). None of the patients who received DOTA-coupled antibody had detectable antibodies against the macrocycle before immunoconjugate administration. We then addressed further the restriction of the immune response against the macrocycle. We found that there was no or very low response against the aromatic ring attached to DOTA. Most, if not all, of the immune response is directed against the DOTA ring structure. Affinity purification of anti-DOTA antibody from serum enabled quantitation of these antibodies in the serum of patients. An inverse, statistically significant correlation was observed between the percentage of binding inhibition of a patient's serum to DOTA, by HSA-2-iminothiolane-DOTA (100 micrograms/ml) and the level of anti-DOTA immunoglobulin in the serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Using linkers to investigate the spatial separation of the conserved nucleotides A9 and G12 in the hammerhead ribozyme.

Two series of hammerhead-derived ribozymes, or 'minizymes', in which helix II has been replaced by linkers of non-nucleotidic moieties, have been synthesised by solid-phase methods. In the first series, the minizymes had linkers containing one, two, three, four or five repeated units of phosphopropanediol, so that the number of atoms in the chain connecting the 3'O of the conserved A9 to the 5'O of the conserved G12 varied from 7 to 31. In the second, more-limited series, the minizymes contained linkers of either tetra- or hexa-ethyleneglycol. The rates at which these minizymes cleaved their cognate 13-nucleotide substrate were determined at 30 degrees C, and compared with the rates of cleavage by an analogous series of minizymes containing from two to six repeated units of thymine deoxyribonucleotide in place of helix II. In all three series, the cleavage rates increased with increasing linker length, with a plateau being reached at the longer lengths tested. Relative cleavage rates within the phosphopropanediol and the thymidine series depended strongly on linker length, but maximal activity was achieved in both series with 25 atoms in the chain joining A9 and G12. The lengths of linkers required to achieve maximal activity of the minizymes are considerably greater than the linkers of 13 atoms which are sufficient to stabilise the ends of double-helices of DNA or RNA.

Structural analysis of a reconstituted DNA containing three histone octamers and histone H5.

Previous work has shown that DNA and the histone proteins will combine to form structures of a complex, yet definite nature. Here, we describe three experiments aimed at a better understanding of the interactions of DNA with the histone octamer and with histone H5. First, there has been some question as to whether the methylation of DNA could influence its folding about the histone octamer. To address this point, we reconstituted the histone octamer onto a 440 base-pair DNA of defined sequence at various levels of cytosine methylation, and also onto the unmethylated DNA. The reconstituted structures were probed by digestion with two different enzymes, micrococcal nuclease and DNase I. All samples were found to contain what appear to be three histone octamers, bound in close proximity on the 440 base-pair DNA. The cutting patterns of micrococcal nuclease and DNase I remain the same in all cases, even if the DNA has been extensively methylated. The results show, therefore, that methylation has little, or no, influence on the folding of this particular DNA about the histone octamer. Second, there has been concern as to whether the base sequence of DNA could determine its folding in a long molecule containing several nucleosomes, just as it does within any single, isolated nucleosome core. In order to deal with this problem, we cut the 440 base-pair DNA into three short fragments, each of nucleosomal length; we reconstituted each separately with the histone octamer; and then we digested the reconstituted complexes with DNase I for comparison with similar data from the intact 440 base-pair molecule. The results show that the folding of this DNA is influenced strongly by its base sequence, both in the three short fragments and in the long molecule. The rotational setting of the DNA within each of the three short fragments is as predicted from a computer algorithm, which measures its homology to 177 known examples of nucleosome core DNA. The rotational setting of the DNA in the 440 base-pair molecule remains the same as in two of the three short fragments, but changes slightly in a third case, apparently because of steric requirements when the nucleosomes pack closely against one another. Finally, there has been little direct evidence of where histone H5 binds within a DNA-octamer complex.(ABSTRACT TRUNCATED AT 400 WORDS)

The intrinsic curvature of DNA in solution.

We propose a detailed quantitative scheme for explaining the anomalous electrophoretic mobility in polyacrylamide gels of repeating sequence DNA. We assume that such DNA adopts a superhelical configuration in these circumstances, and migrates less quickly than straight DNA of the same length because it can only pass through larger holes. The retardation is maximal when the length of the DNA reaches one superhelical turn, but is less for shorter pieces. We attribute the curvature of the superhelix to different angles of roll at each kind of dinucleotide step, i.e. an opening up of an angle by an increased separation on the minor-groove side. The main effect is due to a difference of about 3 degrees in roll values between AA/TT and other steps, together with a difference of about 1 degree in the angle of helical twist: we deduce these values explicitly from some of the available data on gel-running. The scheme involves a simple calculation of the superhelical parameters for any given repeating sequence, and it gives a good correlation with all of the available data. We argue that these same base-step angular parameters are also consistent with observations from X-ray diffraction of crystallized oligomers, and particularly with the recent data on CGCA6GCG from Nelson et al. We are concerned here with the intrinsic curvature of unconstrained DNA, as distinct from the curvature of DNA in association with protein molecules; and this paper represents a first attempt at an absolute determination.

Aircrew dosimetry using the Predictive Code for Aircrew Radiation Exposure (PCAIRE).

During 2003, a portable instrument suite was used to conduct cosmic radiation measurements on 49 jet-altitude flights, which brings the total number of in-flight measurements by this research group to over 160 flights since 1999. From previous measurements, correlations have been developed to allow for the interpolation of the dose-equivalent rate for any global position, altitude and date. The result was a Predictive Code for Aircrew Radiation Exposure (PCAIRE), which has since been improved. This version of the PCAIRE has been validated against the integral route dose measurements made at commercial aircraft altitudes during the 49 flights. On most flights, the code gave predictions that agreed to the measured data (within +/- 25%), providing confidence in the use of PCAIRE to predict aircrew exposure to galactic cosmic radiation. An empirical correlation, based on ground-level neutron monitoring data, has also been developed for the estimation of aircrew exposure from solar energetic particle (SEP) events. This model has been used to determine the significance of SEP exposure on a theoretical jet altitude flight during GLE 42.

Pre-targeted immunoscintigraphy of murine tumors with indium-111-labeled bifunctional haptens.

A method of tumor imaging utilizing the nonspecific accumulation of antibody through leaky capillaries is described, in which the antibody and the radiolabel are administered separately. Nonradioactive antibody is given first (pre-targeted), and allowed adequate time to reach maximum tumor concentration. Depending on the antibody, this may take several days. At the time of maximum tumor concentration of nonradioactive antibody, the blood is quickly cleared of excess circulating nonradioactive antibody using a special i.v. "chase". The radiolabel then is given and imaging done in 1 to 3 hr. The use of short lived tracers (hours) to image antibodies that localize slowly (days) in-vivo is made possible by this method.

Chelate conjugates of monoclonal antibodies for imaging lymphoid structures in the mouse.

Radiolabeling of a mouse monoclonal antibody (MoAb) specific for the mouse histocompatibility alloantigen IAk expressed by the B lymphocytes of BALB/k and C3H mice but not BALB/c mice was performed by mixing the chelate-labeled anti (alpha) IAk MoAb with purified, no-carrier-added 111In citrate. Labeling efficiency was 85-95%, and the labeled alpha IAk MoAb retained its antigen binding properties in vitro and in vivo. The organ, spleen, and lymph node distribution of intravenously and subcutaneously administered 111In alpha IAk MoAb was compared in mice, two IAk positive and one IAk negative strains, and to 125I alpha IAk MoAb in one IAk positive strain. The 111In alpha IAk MoAb was more stable in vivo compared to 125I alpha IAk MoAb, as shown by a much slower excretion and a higher absolute uptake in lymph nodes and spleen. Lymph node to blood ratio was increased twofold by intravenous anti-EDTA MoAb. Subcutaneous injection permitted clear images of the tiny lymph nodes in the mouse. Potential clinical applications of 111In alpha lymphocyte MoAb include localization of normal lymph nodes and T & B cell leukemias and lymphomas, as well as detecting lymphatic metastases of other cancers. Therapy may also be possible using MoAbs labeled with beta-emitting metal ions such as yttrium-90.

Copper-67-labeled monoclonal antibody Lym-1, a potential radiopharmaceutical for cancer therapy: labeling and biodistribution in RAJI tumored mice.

Copper-67 (67Cu) is one of the most promising radiometals for radioimmunotherapy because of its 61.5 hr physical half-life, abundant beta particles, and gamma emissions suitable for imaging. However, 67Cu is readily transferred from the usual chelates of EDTA or DTPA to albumen. We developed a new macrocycle (6-p-nitrobenzyl-TETA) to chelate copper. Bifunctional chelating agent p-bromoacetamidobenzyl-TETA was conjugated to Lym-1, a monoclonal antibody against human B cell lymphoma, without significantly altering its immunoreactivity. This conjugate was stably labeled with 67Cu under conditions chosen to optimize the yield of a high specific activity radiopharmaceutical. The biodistribution in RAJI tumor bearing mice demonstrated significant tumor uptake (14.7% ID per gram) and extended residence time (120 hr) in contrast to normal organs. After 24 hr, radioactivity was continuously cleared from all tissues except the tumor. This study suggests 67Cu labeled Lym-1 to be a promising radiopharmaceutical for potential use for radioimmunotherapy of B cell lymphoma.

Comparative serum stability of radiochelates for antibody radiopharmaceuticals.

Serum incubation of monoclonal antibodies chelate labeled by DTPA, benzyl-EDTA and benzyl-TETA with 111In, 57Co, and 67Cu demonstrated marked differences in their stability. In serum, 111In-benzyl-EDTA-antibody was more stable than 111In-DTPA-antibody. Cobalt-57 or 67Cu chelated antibody were less stable than either 111In chelated antibody; 67Cu was only firmly attached to the antibody as 67Cu-benzyl-TETA-antibody. The relative stability of the radiometal chelated antibodies was paralleled by the relative stability in serum of the radiometal chelates themselves. These in vitro studies suggest that in vivo behavior of metal chelates exposed to a complex protein environment cannot be predicted by classical equilibrium constants.

Metabolism of indium chelates attached to monoclonal antibody: minimal transchelation of indium from benzyl-EDTA chelate in vivo.

The metabolism of radiolabeled antibodies is important for radioimmunoimaging and therapy. The loss of indium-111 (111In) from the chelate can pose problems in imaging and increases the radiation dose to normal tissues. We have evaluated the loss in vivo of 111In from Lym-1-benzyl-EDTA-111In (an antibody conjugated with isothiocyanato-benzyl-EDTA) in normal mice. A monoclonal antibody (CHA 255) that binds to benzyl-EDTA-indium chelates, but not to other forms of indium, was used to measure the percent of 111In remaining in the chelate. Four days after injection, 97.4 +/- 2.2% of the 111In in the liver was still in the benzyl-EDTA chelate, as was 99.4 +/- 0.7% of the 111In in the urine, and 99.1 +/- 0.7% of the 111In in the blood. Studies in vitro indicate that a benzyl-EDTA-111In-antibody-chelate conjugate is more stable in human serum than a benzyl-DTPA-111In conjugate, and that both benzyl-chelate conjugates are much more stable than an unsubstituted DTPA conjugate.

Yttrium-90-labeled monoclonal antibody for therapy: labeling by a new macrocyclic bifunctional chelating agent.

Yttrium-90 (90Y) is a promising radiometal for therapy of cancer due to its high-energy beta emission and a physical half-life of 2.67 days. Bifunctional chelating agents based on DTPA cyclic anhydride or EDTA do not form Y(III) complexes that are stable under physiologic conditions. A new macrocyclic bifunctional chelating agent based on 1,4,7,10-tetraazacylododecane-N,N',N",N"'-tetraacetic acid (DOTA) forms a stable Y(III) complex. It was converted to p-bromoacetamidobenzyl-DOTA (BAD), and conjugated to monoclonal antibody Lym-1 via 2-iminothiolane, either as the free ligand or as the 88Y chelate. Stability studies of Lym-1-2IT-BAD-88Y in human serum in vitro showed no measurable loss of Y(III) from the ligand over a 25-day period. In Raji-tumored mice, tumor uptake was 16.8% of the injected dose per gram of tissue on Day 3. The bone uptake was 2.0, 3.6, and 2.1% injected dose per gram of tissue on Day 1, 3, and 5, respectively. The biodistribution of the control 88Y-citrate demonstrated continuous increase in bone uptake from 13.8% injected dose per gram on Day 1 to 24.9% injected dose per gram on Day 4.

Pretargeted immunoscintigraphy: effect of hapten valency on murine tumor uptake.

A method of radioimmunoscintigraphy using bivalent "Janus" haptens with an apparent enhanced affinity ("avidity") for the antibody is described. Janus with 50 micrograms pretargeted Mab WC3A11 resulted in significantly higher murine tumor concentrations (approximately 7%/g) compared to monovalent haptens (approximately 1.4%/g, p < 0.001), and the same high tumor-to-background ratios (approximately 3/1). Janus was synthesized by coupling two molecules of BABE together with a 1,4 butanedithiol linker. Janus itself was rapidly excreted (T1/2b = 42 min) by the kidneys and did not concentrate in any other organs or tissues. Three-step pretargeted immunoscintigraphy (binder, chaser, tracer) with 111In- or 67Ga-Co(III) Janus produced excellent mouse tumor images in 3 hr with high tumor-to-background ratios. The use of short-lived tracers, such as 99mTc and 68Ga, with a T1/2p of hours to image antibodies that localize slowly over several days in vivo is accessible with this new technology.

Changes in the numbers of retinal ganglion cells and optic nerve axons in the developing albino rabbit.

In albino rabbits aged from the 16th postconceptional day (16PCD) to adulthood, the number of axons in the optic nerves were estimated from sample areas totalling 1-12% of the cross-sectional area of the nerve. On the 16PCD there are about 20,000 axons in the optic stalk. The number of axons in the retrobulbar part of the optic nerve reaches a peak value of 766,000 on the 23PCD, and then decreases to about 350,000 by the 32PCD (the day of birth). The number of axons does not change between the 32PCD and 50PCD, but thereafter it slowly decreases, reaching the adult number (294,000) by the 84PCD. A similar trend is apparent in pigmented animals. Thus, on the 25PCD there are 736,000 axons in the retrobulbar part of the optic nerve and the number decreases to 428,000 by the 31PCD. In the adult pigmented rabbit there are 280,000 axons in the optic nerve. In animals younger than the 32PCD, growth cones are present, and the number of axons in the prechiasmal part of the optic nerve was 8-22% lower than in the retrobulbar part of the same nerve. These observations suggest that there is a continued outgrowth of axons from the eye towards the target nuclei. By the 32PCD, the numbers of axons in the retrobulbar and prechiasmal parts of the nerve were very similar, suggesting that by this age all axons had reached the chiasm. The numbers of retinal ganglion cells (RGCs) labelled by massive injections of horseradish peroxidase into the retino-recipient nuclei were estimated in albino rabbits aged from the 24PCD to adulthood. RGCs were counted in evenly spaced sample areas totalling 4-11% of the retinal area. On the 24PCD, the number of labelled RGCs (500,000) was lower than the number of axons in the optic nerve (probably because not all RGC axons had reached their target nuclei by this age). However, by the 27PCD the number of labelled RGCs (550,000) was very similar to the number of prechiasmal axons (568,000). At all ages thereafter, the numbers of both RGCs and axons were very similar, with adult RGC numbers (about 291,000) being reached by the 85PCD. We conclude that axon loss in the rabbit optic nerve after the 27PCD is almost certainly due to the elimination (presumably death) of the parent RGCs, and we suggest that RGC death is also the most likely cause of axon loss prior to the 27PCD.(ABSTRACT TRUNCATED AT 400 WORDS)

Small, efficient hammerhead ribozymes.

The hammerhead ribozyme is able to cleave RNA in a sequence-specific manner. These ribozymes are usually designed with four basepairs in helix II, and with equal numbers of nucleotides in the 5' and 3' hybridizing arms that bind the RNA substrate on either side of the cleavage site. Here guidelines are given for redesigning the ribozyme so that it is small, but retains efficient cleavage activity. First, the ribozyme may be reduced in size by shortening the 5' arm of the ribozyme to five or six nucleotides; for these ribozymes, cleavage of short substrates is maximal. Second, the internal double-helix of the ribozyme (helix II) may be shortened to one or no basepairs, forming a miniribozyme or minizyme, respectively. The sequence of the shortened helix + loop II greatly affects cleavage rates. With eight or more nucleotides in both the 5' and the 3' arms of a miniribozyme containing an optimized sequence for helix + loop II, cleavage rates of short substrates are greater than for analogous ribozymes possessing a longer helix II. Cleavage of gene-length RNA substrates may be best achieved by miniribozymes.

Differential retinal growth appears to be the primary factor producing the ganglion cell density gradient in the rat.

We have studied the distribution of retinal ganglion cells (RGCs) that had been retrogradely labelled from bilateral injections of horseradish peroxidase into the retino-recipient nuclei of albino rats aged from the 22nd postconceptional day (22PCD-day of birth) to adulthood. During the period in which most (85%) of the naturally occurring RGC loss takes place (22-26 PCD) the distribution of RGCs remains almost uniform. Between the 26 and 32PCD (11th postnatal day), the peak RGC density decreases by only 20% while the RGC density at the superior retinal periphery decreases by 80%. In the same period a centro-peripheral RGC density difference of 4:1 becomes apparent. We have interpreted these changes to be due to a phase of rapid differential retinal growth (with more growth occurring at the retinal periphery). Thereafter the reduction in RGC density (and presumably retinal growth) is more uniform resulting by adulthood in a centro-peripheral RGC density ratio of 5:1.

Metal decomposition rates of 111In-DTPA and EDTA conjugates of monoclonal antibodies in vivo.

We have studied the metal chelate decomposition rates in vivo in both 111In-labelled benzyl EDTA and DTPA (bicyclic anhydride) conjugates of monoclonal anti-IAk IgG2a with identical Ka = 1 X 10(11)M-1 in both Ag+ve and Ag-ve mice. Twenty mu Ci was given i.v. and whole body counting done immediately and daily for 10 days, with six to eight mice in each group. Half the mice in each group received i.p. injections of 5.0 mg CaNa2 EDTA chase (Versenate) to facilitate urinary excretion of free 111In. 50% of control 111In-citrate remained at nine days but only 8% with chase. No significant loss of 111In with chase occurred with C1 substituted EDTA conjugates. A 19% increase in excretion was demonstrated with the chase in mice given DTPA conjugates (1.9% per day). While this will not interfere with radioimmunoimaging up to 24 h after injection, waiting periods of a week or longer will produce significant background of free 111In in the reticuloendothelial system, RES. 111In-EDTA stability was important in accurate metabolic rate measurements of anti-IAk; T1/2 = 7.0 days in Ag-ve mice, T1/2 = 9.3 days in Ag-ve mice. It will be important to measure the in vivo rates for each new metal complex, especially those intended for therapy such as Y-90.

Galactic and solar radiation exposure to aircrew during a solar cycle.

An on-going investigation using a tissue-equivalent proportional counter (TEPC) has been carried out to measure the ambient dose equivalent rate of the cosmic radiation exposure of aircrew during a solar cycle. A semi-empirical model has been derived from these data to allow for the interpolation of the dose rate for any global position. The model has been extended to an altitude of up to 32 km with further measurements made on board aircraft and several balloon flights. The effects of changing solar modulation during the solar cycle are characterised by correlating the dose rate data to different solar potential models. Through integration of the dose-rate function over a great circle flight path or between given waypoints, a Predictive Code for Aircrew Radiation Exposure (PCAIRE) has been further developed for estimation of the route dose from galactic cosmic radiation exposure. This estimate is provided in units of ambient dose equivalent as well as effective dose, based on E/H x (10) scaling functions as determined from transport code calculations with LUIN and FLUKA. This experimentally based treatment has also been compared with the CARI-6 and EPCARD codes that are derived solely from theoretical transport calculations. Using TEPC measurements taken aboard the International Space Station, ground based neutron monitoring, GOES satellite data and transport code analysis, an empirical model has been further proposed for estimation of aircrew exposure during solar particle events. This model has been compared to results obtained during recent solar flare events.

Aircrew exposure from cosmic radiation on commercial airline routes.

As a result of the recent recommendations of the ICRP 60, and in anticipation of possible regulation on occupational exposure of Canadian-based aircrew, an extensive study was carried out by the Royal Military College of Canada over a one-year period to measure the cosmic radiation at commercial jet altitudes. A tissue-equivalent proportional counter was used to measure the ambient total dose equivalent rate on 62 flight routes, resulting in over 20,000 data points at one-minute intervals at various altitudes and geomagnetic latitudes (i.e. which span the full cut-off rigidity of the Earth's magnetic field). These data were then compared to similar experimental work at the Physikalisch Technische Bundesanstalt, using a different suite of equipment, to measure separately the low and high linear energy transfer components of the mixed radiation field, and to predictions with the LUIN transport code. All experimental and theoretical results were in excellent agreement. From these data, a semiempirical model was developed to allow for the interpolation of the dose rate for any global position, altitude and date (i.e. heliocentric potential). Through integration of the dose rate function over a great circle flight path, a computer code was developed to provide an estimate of the total dose equivalent on any route worldwide at any period in the solar cycle.

Thermal environment and sudden infant death syndrome: case-control study.

OBJECTIVE: To compare the thermal environment of infants who died of the sudden infant death syndrome with that of age matched control infants. DESIGN: Case-control study. Infants who died were matched with two controls, one for age and one for age and birth weight. Thermal measurements were conducted at the death scene for cases and at the scene of last sleep for control infants, who were visited unexpectedly within four weeks of the index infant's death on a day of similar climatic conditions. A follow up questionnaire was administered to parents of cases and controls. SETTING: The geographical area served by the professional Tasmanian state ambulance service, which includes 94% of the Tasmanian population. SUBJECTS: 41 infants died of the sudden infant death syndrome at home; thermal observations at death scene were available for 28 (68%), parental questionnaire data were available for 40 (96%). 38 controls matched for age and 41 matched for age and birth weight. RESULTS: Cases had more excess thermal insulation for their given room temperature (2.3 togs) than matched controls (0.6 togs) (p = 0.009). For every excess thermal insulation unit (tog) the relative risk of the sudden infant death syndrome was 1.26 (95% confidence interval 1.05 to 1.52). The average thermal bedding value calculated from parental recall was similar to that observed by attendant ambulance officers (mean difference = 0.4 tog, p = 0.39). Cases were more likely to have been found prone (odds ratio 4.58; 1.48 to 14.11). Prone sleeping position was not a confounder or effect modifier of the relation between excess thermal insulation and the syndrome. CONCLUSIONS: Overheating and the prone sleeping position are independently associated with an increased risk of the sudden infant death syndrome. Further work on infant thermal balance and sudden infant death is required and guidelines for appropriate infant thermal care need to be developed.