The brain-derived neutrophic factor val66met polymorphism and sudden unexpected infant death.

AIM: Findings of hypoxia prior to death and involvement of a dysregulation of the serotonergic network in sudden infant death syndrome (SIDS) may indicate that brain-derived neutrophic factor (BDNF) also is of importance with regard to sudden unexpected infant death. Based on this, the purpose of this study was to investigate the BDNF val66met polymorphism in SIDS cases, cases of infectious death and controls. METHODS: The polymorphism was investigated in 163 SIDS cases, 34 cases of infectious death and 121 controls, using real-time PCR and fluorescence melting curve analysis. RESULTS: There were no differences in val66met genotype distribution between neither the SIDS cases nor the cases of infectious death and controls (p = 0.95 and p = 0.52, respectively). CONCLUSION: The study indicates that the val66met polymorphism is not important for sudden unexpected infant death. However, several other SNPs in the BDNF gene, as well as in other genes involved in this pathway, including G-protein, have to be investigated to fully exclude any involvement of BDNF in SIDS.

Bacteriological investigation--significance of time lapse after death.

In cases of sudden unexpected death in infants and children (SUDI), microbiological investigation has been an important part of the autopsy protocol at the University of Oslo for the last 15 years. The purpose of this study was to compare the microbiological findings in samples taken at hospital admittance shortly after death and at autopsy. Blood cultures and cerebrospinal fluid (CSF) were collected both at the hospital and at autopsy; organ samples were additionally collected at autopsy. Hospital samples were collected at a median of 4.5 h (95% confidence interval [CI] 3.25-5) and autopsy samples at a median of 24.25 h (95% CI 22-25.5) after death. The proportion of positive cultures was stable over time; the post mortal time had no influence on bacterial growth. As long as the autopsy is performed within 48 h after death, prior microbiological examination is unnecessary. Blood culture, CSF and lung specimens are the best predictors in our study.

Determination of cotinine in pericardial fluid and whole blood by liquid chromatography-tandem mass spectrometry.

Cotinine is the main metabolite of nicotine and is used as an indicator of exposure to tobacco smoke. A method has been developed for quantification of cotinine in pericardial fluid and whole blood collected from autopsy casework involving cases of infant death. Sample clean-up was achieved by solid-phase extraction with a mixed-mode column. Cotinine was quantified by liquid chromatography-tandem mass spectrometry. Positive ionization was performed in the multiple reaction monitoring mode. Two transitions were monitored for the analyte and one for the internal standard, cotinine-d(3). The calibration range was 0.9-176 ng/mL for cotinine in both matrixes. The recovery of the analyte ranged from 86 to 92%, and the between-assay precisions ranged from 4 to 6% relative standard deviation. Whole blood and pericardial fluid samples from 95 infant deaths obtained during autopsy were analyzed. A strong correlation (R(2) = 0.97) was found between the cotinine concentrations in pericardial fluid and blood. The correlation was not affected by the postmortem time interval. This study demonstrates that pericardial fluid may be an alternative specimen to blood for quantification of cotinine in forensic autopsies.

The APC gene I1307K variant is rare in Norwegian patients with familial and sporadic colorectal or breast cancer.

Recently, a T-to-A transversion creating an 8-base mononucleotide tract in the APC gene, resulting in substitution of lysine for isoleucine at codon 1307 (I1307K), was found in a subset of Ashkenazi Jews. This sequence variant was most frequent in colorectal cancer patients with a positive family history of colorectal cancer. To determine whether the I1307K variant plays a role in colorectal or breast cancer predisposition in the Norwegian population, we have analyzed blood samples from 210 colorectal cancer patients and 183 breast cancer patients by PCR and direct sequencing. Thirty-seven of the colorectal cancer patients had a positive family history of cancer. Among the breast cancer patients, 24 had a family history of colorectal cancer and 75 a family history of breast and/or ovarian cancer. Only one colorectal cancer patient who belonged to a Jewish family was found to carry the A variant. Our data show that the I1307K variant is rare in the Norwegian population and should not be viewed as a candidate for susceptibility testing for colorectal cancer.

Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history.

Recent reports have suggested that one or more genes may cause replication errors (RER) during colorectal tumorigenesis. Additional alleles are seen in the tumors when analyzing random microsatellite loci. We have studied seven dinucleotide repeat loci, located on seven different chromosomes, by use of polymerase chain reaction amplification and denaturing polyacrylamide gel electrophoresis. We found that 16.5% (40 of 243) colorectal cancers showed RER at one or several loci (RER+). This includes 31% (4 of 13) among cases with a strong positive family history according to previously published criteria and 17% (35 of 207) among cases with no history of familial cancer. Interestingly, no significant association was found between RER+ tumors and a general familial clustering of cancer. Microsatellite instability was significantly associated with DNA diploid status of the tumor (P < 0.001), with the location of the tumor in the proximal colon (P < 0.001), and with poorly differentiated tumor phenotype (P < 0.001). Patients with RER+ at > or = 2 loci tumors had an increased survival (P = 0.05). We further analyzed 84 breast cancers and 86 male germ cell cancers using the same seven markers. None of the tumors were RER+, indicating that this phenomenon may be specific to certain types of tumors.

Estimation of time since death by vitreous humor hypoxanthine, potassium, and ambient temperature.

Measurement of vitreous humor potassium (K(+)) has since the 1960s been recognized as an adjunct for estimation of time since death. In 1991 we introduced hypoxanthine (Hx) as a new marker. Furthermore we demonstrated that time since death estimation was more accurate when ambient temperature was included in the calculations, both for K(+) and for Hx. In this paper we present a refined method. The subjects consist of 132 cases with known time of death and ambient temperature. One sample from each subject was used in the calculations. Vitreous humor Hx levels were available in all subjects, while K(+) was measured in 106 of the subjects, due to insufficient volume of vitreous humor. Linear regression analysis was applied to model the correlation between vitreous humor Hx and K(+), taking the interactions with temperature into consideration. The diagrams published in 1991, which also included ambient temperature, estimated median time since death with range between the 10th and 90th percentile, whereas the linear regression analysis presented in this paper estimates mean time since death with a corresponding 95% interval of confidence. We conclude that time since death may be estimated with relatively high precision applying vitreous humor Hx and K(+) concentrations combined with ambient temperature.

TP53 and long-term prognosis in colorectal cancer: mutations in the L3 zinc-binding domain predict poor survival.

In a consecutive series of 222 colorectal carcinomas from patients with a median follow-up time of 56.8 months (range, 0.5-92.2) treated with surgery, the TP53 gene was screened for mutations. Exons 5-8 were analyzed using constant denaturant gel electrophoresis followed by sequencing, and mutations were found in 102 cases (45.9%). Mutations were found more frequently in rectal tumors versus other locations (P = 0.029) and in aneuploid compared to diploid tumors (P < 0.001). Presence of a TP53 mutation was also significantly associated with absence of microsatellite instability (P = 0.028), as well as with loss of heterozygosity at 17p13 (P < 0.001). The TP53 mutations in the left-sided and rectal tumors were more often transversions than transitions, indicating a different etiology in the development of these tumors. The tendency for shorter cancer-related survival among patients with mutations in their tumors was only statistically significant for patients with left-sided tumors (P = 0.003). All patients with mutations affecting the L3 domain of the protein involved in zinc binding had a significantly shorter cancer-related survival (P = 0.036), indicating that mutations affecting this domain have biological relevance in terms of colorectal cancer disease course. These results suggest that knowledge of a patient's TP53 status, with respect to both the presence and the localization of the mutation, may be important in prognosis evaluation, particularly when selecting patients for more aggressive postoperative therapeutic intervention.

Hypoxanthine levels in vitreous humor: evidence of hypoxia in most infants who died of sudden infant death syndrome.

Postmortem changes of the hypoxanthine in vitreous humor in humans were investigated. Hypoxanthine is formed from hypoxic degradation of adenosine monophosphate. Repeated sampling was performed in 13 deceased adults. Keeping the bodies at +6 degrees C, the increase of the hypoxanthine levels was estimated to 3.5 mumol/L per hour when sampling was started more than 12 hours after death (range 2.8 to 5.6 mumol/L per hour). Results of hypoxanthine measurements from vitreous humor in 73 infants with sudden infant death syndrome, 17 infants and children who died sudden violent deaths, and 6 neonates who died suddenly without hypoxemia prior to death were corrected according to the expected postmortem hypoxanthine increase. The time between death and autopsy was similar in the three groups studied. The corrected median hypoxanthine level in the group with sudden infant death syndrome was 227 mumol/L, which is significantly higher than in the other groups; 22 mumol/L in the group who had violent deaths (P less than .01), and 0 mumol/L in the neonate group (P less than .01). The findings seem to confirm that sudden infant death is preceded by a relatively long period of tissue hypoxia in most cases.

High postmortem levels of hypoxanthine in the vitreous humor of premature babies with respiratory distress syndrome.

To test whether or not premature babies at risk for retinopathy of prematurity have elevated hypoxanthine levels in the eye, the vitreous humor of 13 premature babies who died of severe respiratory distress syndrome and lung failure, was analyzed for hypoxanthine. Their hypoxanthine level was 459 +/- 171 mumol/L (mean +/- SD) compared with 54 +/- 71 mumol/L in seven newborn babies who died suddenly (P less than .001). In 53 adults who died suddenly, the hypoxanthine concentration was 136 +/- 119 mumol/L (P less than .001 when compared with babies with respiratory distress syndrome). Babies with respiratory distress syndrome underwent a significantly longer period with arterial PO2 levels less than 40 mm Hg (5.3 kPa) and they required supplementation with 100% oxygen significantly longer than control babies. The hypoxanthine concentration was correlated with the time during which the arterial PO2 was lower than 40 mm Hg (5.3 kPa) before death, and a significant positive correlation was found (R = .59, P less than .12). The study shows that high levels of hypoxanthine are found in vitreous humor of premature babies with respiratory distress syndrome. Because hypoxanthine is a potential oxygen radical generator and premature babies might have lower levels of antioxidants than full-term babies, it is suggested that the hypoxanthine accumulation in the eyes of premature babies with respiratory distress syndrome could play a pathogenetic role in the development of retinopathy of prematurity.

Elevated levels of hypoxanthine in vitreous humor indicate prolonged cerebral hypoxia in victims of sudden infant death syndrome.

Hypoxanthine levels in vitreous humor from 32 infants who died of sudden infant death syndrome (SIDS) were determined and compared with levels found in eight children who died of trauma, drowning, or hanging and with levels from seven neonates dying suddenly without long-standing antemortem hypoxia. Determination of hypoxanthine level was done with either a PO2 electrode method or high-performance liquid chromatography. The results obtained by both methods were significantly correlated; therefore they were pooled. The median hypoxanthine level in victims of SIDS (380 mumol/L) was significantly higher (P less than .001) than in the children who died violently (118 mumol/L). Moreover, the levels from the SIDS victims were significantly higher (P less than .001) than those from the neonates who died without long-standing hypoxia (53 mumol/L). It is concluded that SIDS is probably not a sudden event but may be preceded by a relatively long period of respiratory failure and hypoxia.

Circulating secretory component in breast neoplasms.

The serum concentrations of IgAp and IgMr associated secretory component (SIgA and SIgM) of 98 patients with neoplasms of the breast were measured. Of the 56 patients with carcinomas, 11 had increased concentrations of circulating SIgM, which was almost twice as sensitive as SIgA as a marker for carcinoma. Concentrations of circulating SIgA and SIgM were independent of expression of secretory component, IgA, and carcinoembryonic antigen (CEA); histological tumour grade; and tumour cell DNA ploidy, whereas a weak correlation between SIgA and SIgM and circulating CEA was seen. The three patients who had liver metastases indicated had particularly high concentrations of circulating SIgA and SIgM, whereas no difference was generally seen between patients with malignancy and those with benign tumours.

Circulating secretory component in relation to early diagnosis and treatment of liver metastasis from colorectal carcinomas.

AIMS: To evaluate serum secretory component in relation to early detection and clinical management of liver metastasis in patients with colorectal cancer. METHODS: Secretory component and carcinoembryonic antigen (CEA) were analysed in serial serum samples from 23 patients who had liver metastases as the only apparent recurrence, and in sera from 54 matched controls. Results of surgical treatment of recurrences were classified peroperatively as radical when no residual tumour was apparent and resection margins were free of disease. RESULTS: In total, 18 (78%) patients had increased secretory component during the whole follow up period (median 16 months); 12 (52%) had raised secretory component concentrations before clinical recurrence (median lead time 5.2 months). There was no difference before recurrence between circulating secretory component and CEA in sensitivity and lead times. Seventeen patients underwent surgery for hepatic metastasis; seven had radical hepatic resection of which only two (29%) showed increased secretory component concentrations before clinical recurrence; both had concurrent raised CEA values. By contrast, secretory component was raised in 83% of those cases considered inoperable. CONCLUSIONS: Although serum secretory component clearly increases in most patients with liver metastases, its clinical value seems questionable because secretory component apparently indicates mainly inoperable hepatic metastases.

Association of p53 accumulation with TP53 mutations, loss of heterozygosity at 17p13, and DNA ploidy status in 273 colorectal carcinomas.

The aim of this study was to establish an experimentally based cutoff level for assessing p53 immunoreactivity in colorectal tumors. The accumulation of p53 protein in 273 colorectal tumors was correlated with previously obtained data on TP53 mutation and loss of heterozygosity at two 17p13 loci in the same tumors. The monoclonal antibody PAb 1801 was used for p53 staining, and the results obtained by immunohistochemistry and immunoblotting were similar. Mutation analyses of exons 5-8 were performed using constant denaturant gel electrophoresis followed by sequencing. There were no statistically significant differences for any measured TP53 gene alteration between the group of tumors without p53-positive nuclei (n = 83) and the group with <5% positive nuclei (n = 58). The majority of mutations within these groups were deletions/insertions and nonsense mutations without p53 accumulation. Therefore, we assume that 5% p53-positive nuclei is the relevant cutoff level to assess TP53 damage in colorectal tumors. A prerequisite for this recommendation is optimal conditions for p53 protein detection. The parameters for p53 dysfunction were correlated to DNA aneuploidy measured by flow cytometry. TP53 mutations were significantly associated with DNA aneuploidy (P < 0.00001), and a nonrandom distribution of TP53 gene alterations among diploid (DI = 1), hyperdiploid (1.0 < DI < 1.3), and highly aneuploid (DI > 1.3) tumors indicates that DNA hyperdiploid tumors constitute a separate developmental entity different from tumors with gross aneuploidy.

Inflammatory responses in sudden infant death syndrome -- past and present views.

Sudden infant death syndrome (SIDS) is sudden unexpected death in infancy for which there is no explanation based on commonly accepted diagnostic criteria; however, half of the victims have had slight signs of infection prior to death. Such slight infection with fever is an important risk factor in combination with a prone sleeping position, especially in infants between 2 and 4 months of age. The purpose of this review is to summarise findings that support the theory that a significant part of cot deaths may be due to an overreaction to otherwise harmless infections. Such factors are mucosal immune stimulation, cytokines in the cerebrospinal fluid and hypoxanthine levels in vitreous humour. The review aims at explaining why we believe that a slight infection combined with a prone position, a warm environment and a vulnerable age period may trigger a vicious circle leading to death.

Comparative epidemiology of sudden infant death syndrome and sudden intrauterine unexplained death.

BACKGROUND: Unexplained antepartum stillbirth and sudden infant death syndrome (SIDS) are major contributors to perinatal and infant mortality in the western world. A relation between them has been suggested. As an equivalent of SIDS, only cases validated by post mortem examination are diagnosed as sudden intrauterine unexplained death (SIUD). OBJECTIVE: To test the hypothesis that SIDS and SIUD have common risk factors. METHODS: Registration comprised all stillbirths in Oslo and all infant deaths in Oslo and the neighbouring county, Akershus, Norway during 1986-1995. Seventy six cases of SIUD and 78 of SIDS were found, along with 582 random controls surviving infancy, all singletons. Odds ratios were obtained by multiple logistic regression analysis. RESULTS: Whereas SIUD was associated with high maternal age, overweight/obesity, smoking, and low education, SIDS was associated with low maternal age, smoking, male sex, multiparity, proteinuria during pregnancy, and fundal height exceeding +2 SD. Thus the effects of maternal age were opposite in SIUD and SIDS (adjusted odds ratio 1.39 (95% confidence interval 1.17 to 1.66) per year, p < 0.0005). Heavy smoking, male sex, and a multiparous mother was less likely in SIUD than in SIDS (0.22 (0.06 to 0.83), 0.22 (0.07 to 0.78), and 0.03 (<0.01 to 0.17) respectively). Overweight/obesity and low fundal height were more common in SIUD than in SIDS (7.45 (1.49 to 37.3) and 13.8 (1.56 to 122) respectively). CONCLUSIONS: The differences in risk factors do not support the hypothesis that SIDS and SIUD have similar determinants in maternal or fetal characteristics detectable by basic antenatal care.

Evaluation of nine different fixatives. 1. Preservation of immunoglobulin isotypes, J chain, and secretory component in human tissues.

Eight cross-linking fixatives were tested for their ability to preserve IgG, IgA, IgM and IgD isotypes, J chain, and secretory component (SC) in paraffin-embedded specimens of human tonsils and colonic mucosa. The results were compared with the antigenic preservation afforded by cold 96% ethanol (with or without inclusion of a prefixation 48-hr washing period). A semiquantitative immunofluorescence scoring system was applied. In relation to the cytoplasmic scores of 3.0 assigned to IgG and IgA immunocytes after ethanol fixation, the following median scores were obtained with the other fixatives: routine formalin, 1.3 (IgG and IgA); glutaraldehyde (1%)-formalin, 0.3 (IgG and IgA); Baker's formol calcium, 2.3 (IgG) and 1.5 (IgA); formol sublimate, 1.0 (IgG) and 1.2 (IgA); acetic acid (2%)-formol saline, 2.0 (IgG and IgA); Bouin's fluid and Susa fixative, 0 (IgG) and 1.3 (IgA); and carbodiimide, 2.3 (IgG) and 2.0 (IgA). Most aldehyde-based fixatives afforded poorer result for IgA immunocytes when an anti-alpha-chain reagent of restricted specificity was applied. The result was usually slightly better for IgM cells than for IgG cells, but IgM did not resist fixation in Bouin's fluid and Susa fixative to the same extent as IgA. IgD immunocytes were poorly revealed except after fixation with Baker's formol calcium, carbodiimide, and acetic acid-formol saline, which afforded median fluorescence scores of 1.0-1.8 compared with 3.0 after ethanol fixation. J chain in IgA immunocytes was most intensely stained in sections of ethanol-fixed tissue denatured in acid urea (median score, 3) but was also well revealed after fixation with formalin, acetic acid-formol saline, Bouin's fluid or Susa fixative (median scores, 1.8-2.0). The results with most of the cross-linking fixatives were much less favorable for IgA, IgM, J chain and SC in colonic crypt epithelium than in immunocytes. Only carbodiimide afforded fluorescence scores for IgA and SC approaching those obtained after ethanol fixation (2.0 and 1.5 vs. 3.0 and 2.8). The effect of proteolytic unmasking depended on the fixative - not only for different antigens and locations in the tissue, but also for the same antigen in apparently similar cells.

Immunohistochemical study of secretory component, secretory IgA and carcinoembryonic antigen in large bowel carcinomas.

The epithelium of 41 large bowel carcinomas was scored immunohistochemically on a semiquantitative basis for the presence of secretory component (SC), secretory IgA, and carcinoembryonic antigen (CEA). Both the tumour and the adjacent "transitional mucosa" were evaluated. The various immunofluorescence scores obtained, the histological grades of the tumours, their Dukes' stages, and the plasma CEA levels were subjected to non-parametric correlation analyses. Tumour SC was positively correlated with histological tumour grade and inversely related to Dukes' stage. Tumour secretory IgA generally showed a pattern similar to that of SC. Tumour CEA showed no correlation with any of the other parameters. The contents of SC and secretory IgA in the transitional mucosa were negatively correlated with Dukes' stage and plasma CEA. Whether the variations observed in the epithelial cell markers reflected primary events in the malignant development or secondary alterations is unknown. Nevertheless, especially the amount of tumour SC may turn out to be of prognostic value.

Survival time and acting capability after fatal injury by sharp weapons.

Autopsy reports from 109 victims of fatal injury from sharp weapons were investigated with regard to type and number of lesions, blood loss, degree of alcohol intoxication, survival time and acting capability after the injury. Of the 13 who died immediately, nine had penetrating lesions of the heart. This group also had the highest number of lesions. Sixty-four victims survived for some time. The survival time increased with decreasing number of lesions. The greatest blood loss and the highest blood alcohol concentrations were found in those who survived between 0.5 h and 1 h. Twenty-four victims were able to make physical efforts after the injury and the movements varied from a few steps to the running of several hundred meters. Decisive factors for decrease in survival time and acting capability are penetrating lesions to the heart and the great vessels, and multiplicity of injuries.

SIDS--changes in the epidemiological pattern in eastern Norway 1984-1996.

The objective was to analyse differences in the epidemiological pattern of sudden death in infancy during two time periods--the Sudden Infant Death Syndrome (SIDS) 'epidemic': 1984-1989, and the period of rapid decline in the SIDS rate 1990-1996. Sex distribution, age, sleeping position, signs of infection, day of the week and place of death were registered and compared for the two time periods studied in all SIDS cases autopsied at the Institute of Forensic Medicine, Oslo. There were significantly more deaths in the age group under four months in the period 1984-89 than in the second period. Prone sleeping position, signs of infection, death outdoors and during the winter were more frequent during the first period than in the second. These features also were more frequent in the age group under four months than in the older babies during the first period. The shift in the epidemiological pattern after 1990, when the risk factor campaign was launched, indicates that prone sleeping position, cold climate, sleeping outdoors and infections seem to be risk factors that are particularly harmful to the youngest infants.

A new biochemical method for estimation of postmortem time.

Hypoxanthine (Hx) is formed by hypoxic degradation of adenosine monophosphate (AMP) and might be elevated due to antemortem hypoxia. However, it also increases after cessation of the life processes. Until now measurements of potassium in corpus vitreous humor have been used by forensic pathologists to determine postmortem time. In this study the influence of postmortem time and temperature on vitreous humor Hx and potassium levels were compared. Repeated sampling of vitreous humor was performed in 87 subjects with known time of death and diagnosis. The bodies were kept at either 5 degrees C, 10 degrees C, 15 degrees C or 23 degrees C. Hx was measured by means of HPLC and potassium by flame photometry. In 19 subjects from whom samples were obtained within 1.5 h after death, the normal level of Hx could be estimated to be 7.6 mumol/l and that of potassium to be 5.8 mmol/l. The spread of the potassium levels measured shortly after death was much greater than for the corresponding Hx levels. In the four temperature groups the Hx level increased 4.2, 5.1, 6.2 and 8.8 mumol/l per h, respectively, whereas the corresponding figures for potassium were 0.17, 0.20, 0.25 and 0.30 mmol/l per h. The vitreous humor concentration of both Hx and potassium increases fairly linearly after death. The slopes are steeper with increasing temperature. Since the scatter of the levels is greater for potassium than for Hx, the latter parameter seems to be better suited for the determination of time of death in cases without antemortem hypoxia, especially during the first 24 h.