Fungal encephalitis following bone marrow transplantation: clinical findings and prognosis.

BACKGROUND: Central nervous system fungal infections (FI) are important complications and a cause of mortality in patients who receive hematopoietic stem cell transplantation (HSCT). AIMS: To study the clinical aspects of fungal encephalitis (FE). SETTINGS AND DESIGN: The study was carried out at the HSCT Center of the Hospital de Clínicas, Federal University of Paraná, Curitiba, Brazil. MATERIALS AND METHODS: Clinical records and autopsy reports from patients submitted to HSCT with a diagnosis of FE. RESULTS: Twelve patients were diagnosed with FE presenting with lowered level of consciousness, hemiparesis and seizures. We were able to identify two subgroups regarding susceptibility to FE: (1) patients with early onset FI and severe leucopenia, and (2) patients with later onset FI with graft-versus-host disease using immunosuppressive drugs. Eleven of the patients died directly due to the neurological complication, all had post-mortem confirmation of the diagnosis of FI. CONCLUSIONS: These clinical, paraclinical and temporal patterns may provide the opportunity for earlier diagnosis and interventions.

Scab diameters on pig skin resulting from hot particle exposures under varying conditions.

The National Council on Radiation Protection and Measurements (NCRP) in NCRP Report Number 106 recommended a limit of 10(10) beta particles emitted from radioactive particles with sizes less than 1 mm (hot particles) to prevent acute deep ulceration. This recommendation was determined, in part, by regressing the diameter of the scabs induced by fissioned 235UC2 hot particles as a function of the logarithm of the number of beta particles emitted from the sources for one study. To validate this recommendation and the approach used by the NCRP, external irradiations of pig skin using radioactive sources of less than 600 microm in the largest dimension were carried out. The hot particles used included fissioned 235UC2 and activated 170Tm, 175Yb, and 46Sc. Results indicated a strong correlation between scab diameter and dose for scabs induced using fissioned 235UC2, activated 170Tm, and 46Sc, but not for 175Yb. The correlation value decreased with decreasing beta particle energy, with the exception of 46Sc, which had scabs with diameters greater than twice the maximum beta particle range. The larger scabs from 46Sc are thought to be due to dose contributions from the gamma rays. The results also give an ulceration threshold less than that given by NCRP to prevent acute deep ulceration. It was concluded that regression analysis of scab diameter as a function of either number of beta particles emitted from the hot particles or dose did not yield either precise or accurate thresholds but was useful in determining probable ranges of doses which lead to scab induction.

Scab incidence on pig skin resulting from hot particle exposures under varying conditions.

External irradiations of pig skin using radioactive sources of less than 600 microm in the largest dimension (hot particles) were carried out. The objective of the study was to determine a threshold for scab induction. Hot particles used included fissioned 235U and activated 170Tm, 17SYb, and 46Sc with maximum beta-particle energies of approximately 1.8 (average), 0.97, 0.47, and 0.35 MeV, respectively. The photon emissions from the fissioned 235U were about 1 MeV per disintegration. The photon emissions from 46Sc were 0.89 and 1.12 MeV, with 100% abundance. Photon emissions from 170Tm and 175Yb were negligible. Responses followed cumulative normal probability distributions; thus, no true thresholds could be determined. Hence, 10% and 50% scab incidence rates (ED10 and ED50, respectively) were determined using probit analysis. For dose averaged over 1 cm2 at a depth of 70 microm, the ED10 (and 95% confidence limits) were 5.1 (2.7-7.2) Gy for 46Sc, 1.3 (0.89-1.8) Gy for 175Yb, 2.8 (1.9-3.5) Gy for 170Tm, 8.5 (5.7-9.8) Gy for on-skin fissioned UC2, and 4.5 (0.9-7.2) Gy for off-skin fissioned UC2. The ED50 values were 12 (8.8-17) Gy for 46Sc, 6.0 (4.3-9.7) Gy for 175Yb, 5.9 (5.1-6.7) Gy for 170Tm, 11 (9.9-19) Gy for on-skin fissioned 235UC2, and 11 (6.2-14) Gy for off-skin fissioned 235UC2.

Effective half-lives for patients administered radiolabeled antibodies and calculated dose to the public in close proximity to patients.

Effective half-lives (T(e)) for radiolabeled antibodies can be much longer than that of traditional radiopharmaceuticals, potentially resulting in larger doses to members of the public. Clearance-rate data from patients treated with radio-labeled antibodies (RABs) were obtained from ten institutions. Calculations were made to determine if a single- or bi-exponential clearance-rate model was statistically justified; the results indicated that the former model was justified for more than 95% of the data. Values of T(e) for the different RABs are summarized. To plan actions to limit doses to less than 5 mSv annually for individuals continuously close to the patient (at 1 m), dose rates from patients at the time of release also are given as a function of T(e).

Tritium radiobiology and relative biological effectiveness.

During the past decade, a large number of radiobiological studies have become available for tritium--many of them focusing on the relative biological effectiveness of tritium beta rays. These and previous studies indicate that tritium in body water produces the same spectrum of radiogenic effects (e.g., cancer, genetic effects, developmental abnormalities, and reproductive effects) observed following whole-body exposure to penetrating radiations such as gamma rays and x rays. However, tritium beta rays are of greater biological effectiveness than gamma rays and x rays. For example, tritium in the oxide form is about 2 to 3 times more effective at low doses or low dose rates than gamma rays from 137Cs or 60Co. When tritium is bound to organic molecules, relative biological effectiveness values may be somewhat larger than those for tritium in oxide form. Tritium administered to animals or to cells in vitro as tritiated amino acids results in relative biological effectiveness values that appear similar to those obtained for tritium in oxide form; however, if administered as tritiated thymidine, the relative biological effectiveness values appear to be about two-fold higher. It is clear from the wealth of tritium data now available that relative biological effectiveness values for tritium beta rays are higher than the quality factor of unity generally used in radiation protection.

Severity of organ injury as a predictor of acute mortality for disparate patterns of absorbed dose distribution.

Nonuniform distribution of absorbed dose is frequently encountered in the irradiated mammal; the degree of nonuniform distribution is generally more severe as the size of the animal increases and the energy or penetrating power of the radiation decreases. However, acute mortality under these conditions, e.g., from the hematopoietic syndrome, appears not to be consistently predictable from the dose at any given location or locations within the animal. It is thus reasonable to seek a biological quantity that may be adequate for this purpose. Accordingly, it was postulated that, in animals dying from the bone marrow syndrome, survival is determined by the total number of viable stem cells remaining in the entire body, independent of their distribution. To test this hypothesis, the LD50/30 value for mice exposed to nonuniform irradiation of varying degrees of severity was obtained, as was that for mice receiving uniform total-body irradiation. The distribution of bone marrow in transverse segments of tissue along the spinal axis was determined, as was the dose to each of the segments. The data were analyzed by multiplying, for each segment, the fraction of stem cells in the fraction of cells surviving, as determined from the dose and a survival curve for stem cells determined separately. The sum of these products yielded the surviving number of stem cells in the total mouse, for both the uniformly and nonuniformly exposed animals. The surviving fraction was found to differ by no more than 20%; this was taken to be reasonable evidence that, based on the number of surviving stem cells, it is possible to predict the mortality rate for both uniform and markedly nonuniform irradiation.

A system for simultaneous exposure of small animals to 60-Hz electric and magnetic fields.

Equipment designed for simultaneous exposure of rodents to 60-Hz electric and magnetic fields is described. Three identical systems were constructed, each capable of continuous exposure of 256 rats or 640 mice to a nominal electric field at less than 50 kV/m, and to horizontal and vertical magnetic fields at less than 1 mT. Design features, construction details, and results of various tests of the systems are described. Tests were made: of phase relations between electric and magnetic fields; of uniformity of electric and magnetic fields; of changes across time in electric-field intensity as a result of animals' soiling of cages and various washing routines; of resistance of bedding material during humid and dry conditions; and of acoustic noise due to background, to field-generation equipment, and to air conditioning equipment. The results demonstrated that fields were effectively generated but that significant and troublesome changes in electric-field intensity occurred because of cage-soiling. However, when cages were frequently cleaned, field intensities were consistent from one exposure to another.

Cytogenetic analysis of mouse metaphase II oocytes following exposure to tritiated water.

Female mice were given different dosages (0, 3.0, 7.5, 15.0, or 30 muCi/ml) of tritium in their drinking water continuously from 3 to 7 weeks of age to assess the effects on germ cell chromosomes. At 8-9 weeks of age, mice were superovulated and metaphase II oocytes were processed and C-banded for cytogenetic analyses. Chromatid acentric fragments were the only type of structural aberration detected, and their incidence was higher in controls than in any of the tritiated water (HTO) groups. Analysis of numerical chromosomal aberrations revealed that the incidence of hypoploid (N = 19) oocytes was higher in oocytes from mice who drank HTO as compared with controls. However, the effects of HTO upon aneuploidy induction was not definitive due to the increase the incidence of aberrations in mouse oocytes can be related to the low dose rate resulting from chronic HTO exposure and possibly death of tritium-damaged cells.

Effects of low level radiation upon the hematopoietic stem cell: implications for leukemogenesis.

These studies have addressed firstly the effect of single small doses of x-rays upon murine hematopoietic stem cells to obtain a better estimate of the Dq. It is small, of the order of 20 rad. Secondly, a dose fractionation schedule that does not kill or perturb the kinetics of hemopoietic cell proliferation was sought in order to investigate the leukemogenic potential of low level radiation upon an unperturbed hemopoietic system. Doses used by others in past radiation leukemogenesis studies clearly perturb hemopoiesis and kill a detectable fraction of stem cells. The studies reported herein show that 1.25 rad every day decrease the CFU-S content of bone marrow by the time 80 rads are accumulated. Higher daily doses as used in published studies on radiation leukemogenesis produce greater effects. Studies on the effect of 0.5, 1.0, 2.0, and 3.0 rad 3 times per week are under way. Two rad 3 times per week produced a modest decrease in CFU-S content of bone marrow after an accumulation of 68 rad. With 3.0 rad 3 times per week an accumulation of 102 rad produced a significant decrease in CFU-S content of bone marrow. Dose fractionation at 0.5 and 1.0 rad 3 times per week has not produced a CFU-S depression after accumulation of 17 and 34 rad. Radiation leukemogenesis studies published to date have utilized single doses and chronic exposure schedules that probably have significantly perturbed the kinetics of hematopoietic stem cells. Whether radiation will produce leukemia in animal models with dose schedules that do not perturb kinetics of hematopoietic stem cells remains to be seen.

The tritium toxicity program in the Medical Department at Brookhaven National Laboratory.

A summary of all the findings to date is given in Table 2. It appears from this information that it is possible to detect somatic, cytogenetic, and genetic effects resulting from exposures at 33 to 100 times the mpc's for HTO. Similar effects also result from exposure to external gamma rays at an equivalent dose. The reduction in bone marrow cells in animals maintaining normal total cellularity is of interest since it demonstrates both the presence of an effect at the primitive cell level and the animal's ability to compensate for this effect by recruiting stem cells from the G0 resting state. This evidence of damage together with the observed cytogenetic changes leads one to contemplate the possible importance of radiation exposures at these levels for the induction of leukemia or other blood dyscrasias. Studies to investigate this question are now under way. As predicted on the basis of established principles of radiobiology, exposure to tritium beta rays from HTO ingestion results in measurable effects on several animal systems. The importance of position of incorporation of H into molecules of biological importance has not been well defined, nor have the low-dose portions of the dose-response curve for several effects of interest. Experiments designed to address these questions and measure H turnover as a means for analysis of cell kinetics in several systems are now under way.

Sister chromatid exchanges in bone marrow cells of mice maintained on tritiated water.

The ability of tritium to induce sister chromatid exchanges (SCEs) has been investigated in male mice of the Hale-Stoner-Brookhaven strain maintained on drinking water containing 3.0 microCi/ml tritiated water (HTO). At selected intervals after 28-261 days of consuming HTO, the frequency of SCEs and the kinetics of cellular proliferation were measured in bone marrow cells of animals maintained on HTO, and in age-matched control groups, by 5-bromo-2'-deoxyuridine labelling methods. A statistically significant (1 percent level) elevation of SCEs was observed after 81, 163, 192, 247 and 261 days of HTO ingestion. The frequency of induced SCEs increased linearly with the ingestion time. These results are of particular interest since ionizing radiation is generally not considered to be an efficient inducer of SCEs.

Residual toxicity in hematopoietic cells following a single dose of methylnitrosourea.

The residual injury to the proliferation capability of hemopoietic stem cells (CFU-S) which results from their exposure to leukemogenic agents was evaluated in mice given a single leukemogenic dose of methyl nitrosourea (MNU 50 mg/kg body weight, i.v.). Bone marrow cellularity, splenic weight, number of CFU-S and the proportion of cycling to noncycling CFU-S were measured in an effort to detect and acute and residual injury to the CFU-S from mice given MNU 21 and 3 days earlier. Marrow cells were also transferred into lethally irradiated mice to observe the self-renewal capability of the CFU-S in the recipient spleen and bone marrow. The results of these measurements show that the CFU-S in marrow from mice given 50 mg/kg of MNU 21 days earlier still have a defective ability for self-renewal, although the total cellularity, number of CFU-S and proportion of cycling and noncycling CFU-S in the donor have returned to the normal range. The relationship of this self-renewal defect to the development of leukemia after this leukemogenic dose of MNU is not known.

The distribution of tritium among the amino acids of proteins obtained from mice exposed to tritiated water.

The distribution of tritium among the amino acids of serum proteins in mice chronically exposed to tritiated water was determined by ion exchange chromatography of the protein hydrolysate. The specific activity of nonexchangeable tritium in these amino acids relative to the specific activity of tritium in the tissue water of mice ranged from 0.04 for phenylalanine and threonine to 1.0 for glycine and alanine. Since tritium from tissue water can enter the nonexchangeable positions of amino acids only as the result of metabolic processing, the relative specific activity of tritium in each amino acid is an indicator of the extent of such processing. The tritium content of tyrosine and all the amino acids required in the diet for survival is quite low, except for histidine, and can be entirely accounted for by transamination or, in the case of methionine, by transmethylation. The tritium content of the other amino acids is too high to result from such minor processing and must reflect primarily the fraction synthesized de novo. The implications of these findings with respect to the radiobiological consequences of a diet containing tritiated proteins are discussed.

Effects of benzene inhalation on murine pluripotent stem cells.

Effects of benzene inhalation on mouse pluripotent hematopoietic stem cells have been evaluated. Male mice 8--12 wk old were exposed to 400 ppm benzene for 6 h/d, 5 d/wk, for up to 9 1/2 wk. At various time intervals exposed and control animals were killed, and cardiac blood was evaluated for changes in white blood cell (WBC) and red blood cell (RBC) content. In addition, femora and tibiae were evaluated for total marrow cellularity, stem cell content (as measured by the spleen colony technique), and the percent of stem cells in DNA synthesis (as determined by the tritiated thymidine cytocide technique). Exogenous spleen colonies grown from marrow of exposed animals were counted, identified, and scored by histological type. Exposure to benzene caused significant depressions of RBCs and WBCs throughout the exposure period, which continued for at least 14 d after exposure. Bone marrow cellularity and stem cell content were also depressed in exposed animals throughout the study. Tritiated thymidine cytocide of spleen colony-forming cells was generally increased in exposed animals, perhaps indicating a compensatory response to the reduction of circulating cells. Spleen colonies of all types were depressed after exposure to benzene. The significance of the reduction in cellularity, stem cell content, and changes in morphology of spleen colonies is discussed in relation to cellular toxicity and residual injury.

Histone turnover within nonproliferating cells.

The turnover of DNA and histones in the livers and brains of mice has been determined. These mice had been exposed to constant levels of tritiated water from conception until they were 8 months old. At this point, exposure to tritium was discontinued, and the tritium remaining in DNA and histones was measured at various intervals afterward. The half-lives calculated for these components (with 95% confidence limits given in parentheses) were 117 (85-188) days for liver histone, 318 (241-466) days for liver DNA, 159 (129-208) days for brain histone and 593 (376-1406) days for brain DNA. The difference between histone and DNA turnover is statistically significant for both tissues and indicates that histone turnover within tissues cannot be solely accounted for by cell turnover within the tissue but also must include histone turnover within living cells. The half-life of histone within cells is estimated to be 117 (88-178) days in liver and 223 (187-277) days in brain.

Separation and concentration of murine hematopoietic stem cells (CFUS) using a combination of density gradient sedimentation and counterflow centrifugal elutriation.

To obtain concentrated suspensions of pluripotent hematopoietic stem cells *CFUS) from murine bone marrow, density gradient centrifugal sedimentation (DGCS) was combined with counterflow centrifugal elutriation (CE). This combination provided a 7.6 fold enrichment of the CFUS concentration. For DGCS, Percoll a suspension of silica particles coated with polyvinylpyrrolidone was used. For fractionation by the CE an elutriator rotor (JE-6, Beckman) was used for further concentration of the cells harvested from the DGCS. Bone marrow erythropoiesis was suppressed by transfusion plethora initiated 5-6 days before the bone marrow was harvested. These two physical separation procedures combined with transfusion plethora to suppress erythropoiesis are effective in producing an enriched fraction of CFUS without change in distribution of the histologic type of colonies.

Kinetics of proliferation and differentiation of human hemopoietic cells in a diffusion chamber system.

Proliferation and differentiation of human granulocytes were studied in a diffusion chamber (DC) culture system. Evaluation of the nucleated marrow cells of normal volunteers revealed that the granulocytic precursors continue differentiation in the DC as they do in vivo. Generation time of the granulocytes in the multiplicative pool in DC was determined by the autoradiographic mean grain count halving method. With tritiated thymidine (3HTdR) the half-time was 67 h, where as with 125Iododeoxyuridine it was 47 h. The longer half-time with 3HTdR supports the notion that a fraction of nuclear 3HTdR from dead cells is reutilized by the newly proliferating cells. The 3HTdR labeling index of granulocytic precursors in the multiplicative pool during the first 10 days in DC is not constant, suggesting a variation in the rate of proliferation. The DNA synthesis time in DC, determined by double isotope labeling technique, showed that it was relatively constant. Simultaneous evaluation of rate of transition of granulocytes in DC from one stage to the next of one patient with chronic myelocytic leukemia and another with myeloid metaplasia with myelofibrosis indicated a more rapid transition in the DC than in vivo in man.