Sudden infant death following hexavalent vaccination: a neuropathologic study.

We examined a large number of sudden infant death syndrome victims in order to point out a possible causal relationship between a previous hexavalent vaccination and the sudden infant death. We selected 110 cases submitted to in-depth histological examination of the autonomic nervous system and provided with detailed clinical and environmental information. In 13 cases (11.8%) the death occurred in temporal association with administration of the hexavalent vaccine (from 1 to 7 days). In none of these victims congenital developmental alterations of the main nervous structures regulating the vital functions were observed. Only the hypoplasia of the arcuate nucleus was present in 5 cases. In one case in particular an acquired hyperacute encephalitis of the tractus solitarii nucleus was diagnosed in the brainstem. This study does not prove a causal relationship between the hexavalent vaccination and SIDS. However, we hypothesize that vaccine components could have a direct role in sparking off a lethal outcome in vulnerable babies. In conclusion, we sustain the need that deaths occurring in a short space of time after hexavalent vaccination are appropriately investigated and submitted to a post-mortem examination particularly of the autonomic nervous system by an expert pathologist to objectively evaluate the possible causative role of the vaccine in SIDS.

Feto-placental atherosclerotic lesions in intrauterine fetal demise: role of parental cigarette smoking.

The atherogenic effect of cigarette smoking is already recognizable in coronary arteries of fetuses in the last gestational weeks. In this study we analyzed the atherogenic effect of mother's and father's smoking habit on coronary arteries and even on adnexa of 30 human fresh fetuses died from 32 to 41 gestational weeks. In 12 cases only the mothers of the victims were cigarette smokers, in 7 cases only the fathers were smokers, whereas in 11 cases nobody smoked.We observed pre-atherosclerotic and initial atherosclerotic lesions of the adnexa in 21 cases, of which 11 cases had only mother smokers and 6 cases only father smokers. The atherogenic effect is statistically significant in both smoker groups, but stronger in maternal one. The atherosclerotic lesions found in umbilical and placental arteries are similar to those described in fetal coronary arteries: thickening of the arterial walls caused by proliferation and migration of the smooth muscle cells of the tunica media with loss of polarity and infiltration of the subendothelial connective tissue.

Hypoplasia and neuronal immaturity of the hypoglossal nucleus in sudden infant death.

OBJECTIVE: To report the case of five month old female baby with a history of episodes of gastro-oesophageal reflux and pneumonia. Her sudden death offered a unique insight into the possible role of delayed neuronal maturation and hypoplasia of the hypoglossal nucleus in representing a likely morphological substrate of sudden death. METHODS: Morphometric analysis was carried out with an Image-Pro Plus Image analyser (Media Cybernetics) on both sides of the brain stem. RESULTS: Hypoplasia and neuronal immaturity of the hypoglossal nucleus were demonstrated, accompanied by hypoplasia of the arcuate nucleus. CONCLUSIONS: Much attention should be paid to the possible role of the hypoglossal nucleus in determining a lethal outcome in infancy through impairment of deglutition and subsequent recurrent episodes of pneumonia, and as a necropsy finding.

Sudden infant death triggered by dive reflex.

The dive reflex is the reflex mechanism most frequently considered in the aetiopathogenesis of sudden infant death syndrome (SIDS). This seems to persist in human beings as an inheritance from diver birds and amphibians. It has been reported that washing the face with cold water or plunging into cold water can provoke cardiac deceleration through the intervention of the ambiguus and the vagal dorsal nuclei. This report describes a case of SIDS that offers a unique insight into the role of the dive reflex in determining a lethal outcome. Examination of the brainstem on serial sections revealed severe bilateral hypoplasia of the arcuate nucleus and gliosis of the other cardiorespiratory medullary nuclei. The coronary and cardiac conduction arteries presented early atherosclerotic lesions. The possible role of parental cigarette smoking in the pathogenesis of arcuate nucleus hypoplasia and early coronary atherosclerotic lesions is also discussed.

Role of somatostatin and apoptosis in breathing control in sudden perinatal and infant unexplained death.

OBJECTIVES: To obtain basic information about the expression of somatostatin in the human central nervous system and, in particular, to evaluate its possible involvement in unexplained perinatal and in sudden infant death syndrome. MATERIAL: Sixty-seven brainstems from subjects aged from 30 gestational weeks to 12 postnatal months, dying of both known and unknown causes, were selected for this study. The unexplained deaths included 17 sudden intrauterine deaths, 5 sudden neonatal deaths and 28 sudden infant deaths. METHOD: All brainstems were fixed in 10% phosphate-buffered formalin, processed and embedded in paraffin, according to our protocol available on the web site: http://users.unimi.it/-pathol/sids/riscontro_diagnostico_e.html. The distribution of the somatostatin in the brainstem was studied by immunohistochemistry on serial sections. RESULTS: We observed an intense somatostatin positivity in many brainstem nuclei prevalently involved in the respiratory activity (parabrachial/Kölliker-Fuse complex, locus coeruleus, hypoglossus nucleus, dorsal vagus motor nucleus, tractus solitarii nucleus, ambiguus nucleus, reticular formation) in stillbirths. In 10 fetuses with unexplained death the neurons of the hypoglossus nucleus were somatostatin-negative. In the postnatal deaths, we observed immunopositivity in the ventrolateral and ventral subnuclei of the tractus solitarii nucleus. Besides, in 15 sudden infant death victims and in 1 control case, somatostatin-positive neurons were also present in the hypoglossus nucleus. In 10 of these 15 cases, a high apoptotic index was also reported. CONCLUSIONS: We suggest that abnormalities in the distribution of SS in the hypoglossus nucleus before and after birth may contribute to the induction of both fatal breathing in prenatal life and abnormal ventilatory control after birth leading to irreversible apnea.

Autoptic examination in sudden infant death syndrome and sudden intrauterine unexpected death: proposal of a national law.

The autoptic procedures in sudden infant death syndrome (SIDS) and sudden intrauterine unexpected death (SIUD) victims requires an in-depth examination of the autonomic nervous system, central and peripheral, as well as of the cardiac conduction system. In these pathologies, our studies have identified frequent developmental abnormalities in the brain stem, particularly hypoplasia of the arcuate nucleus, and accessory atrioventricular pathways in the cardiac conduction system. The law 'Post-mortem Investigation on the SIDS and of Unexplained Late Fetal Death' designated the Institute of Pathology of Milan University as the National Reference Center. The objectives of this law include, in article 4, widespread informative and preventive campaigns and continuous formation of programs for medical personnel.

Intimal preatherosclerotic thickening of the coronary arteries in human fetuses of smoker mothers.

BACKGROUND: Many studies have described the development of preatherosclerotic coronary artery lesions in infancy. The observations reported in the literature regarding the fetal origin of coronary artery lesions are rare and controversial. OBJECTIVES: To identify the features of preatherosclerotic coronary artery lesions in late fetal stillborns and the possible atherogenic role of maternal cigarette smoking. METHODS; We examined 22 stillborns (13 males and nine females), all of whom had died sine causa after the 32nd week of gestation. All underwent autopsy. Twelve of the mothers smoked over five cigarettes per day before and during the pregnancy. The four major epicardial coronary arteries were isolated along their whole length, embedded in paraffin and serially cut for histologic examination and immunohistochemical studies, particularly searching for the proliferating cell nuclear antigen and c-Fos expression. Alterations of chromosome 7 were also investigated by the fluorescence in situ hybridization technique. RESULTS: In over 50% of the fetuses, almost all from smoker mothers, multifocal structural alterations of coronary walls were evident. The smooth muscle cells (SMCs) presented loss of polarity, forming columns perpendicular to the axis of the media and infiltrating the subendothelial connective tissue. Increased amounts of mucoid ground substance were also observed in the subendothelial connective tissue. In all the cases with coronary alterations, study of the biological markers showed intense c-Fos positivity of the SMCs. CONCLUSIONS: Preatherosclerotic intimal alterations of the coronary arteries are already detectable in the prenatal period and are significantly associated with maternal cigarette smoking.

Brain stem lesions in the sudden infant death syndrome: variability in the hypoplasia of the arcuate nucleus.

In the present study we investigated quantitatively the incidence of hypoplasia of the arcuate nucleus (ARCn) of the medulla oblongata, reported earlier [Gozal D, Hathout GM, Kirlew KAT (1994) J Appl Physiol 76:207], as well as its distribution in 62 cases of sudden infant death syndrome (SIDS; mean age 14 postnatal weeks, 39 male and 23 female) and 25 controls (mean age 16 postnatal weeks, 14 male and 11 female), using detailed histopathological and morphometric analyses performed on serial sections of medulla oblongata. The SIDS cases were divided into four subtypes: SIDS A (27 cases, 43%) with histologically well-developed ARCn; SIDS B (16 cases, 26%) with severe bilateral hypoplasia along the whole length; SIDS C (11 cases, 18%) with partial bilateral hypoplasia, located mainly in the lateral portions of the caudal two thirds of the nucleus, and SIDS D (8 cases, 13%) with right monolateral hypoplasia of the ARCn. ARCn hypoplasia was detected in 56% of cases (35 cases). Three-dimensional volume reconstruction showed that in the SIDS A victims the mean volume was analogous to controls, whereas in the SIDS group with ARCn hypoplasia, severe or partial, the mean volume was significantly different from controls on both sides of the medulla oblongata (SIDS B group: P=0.003, P=0.002; SIDS C group: P=0.007, P=0.008). The mean ARCn volume in the SIDS D group was statistically significant only on the right side ( P=0.005). We also observed reduced neuron density of the ARCn, associated with a decrease in the total number of neurons over the whole length of the nucleus itself. On the basis of the morphometric results of neuronal population in the different portions of the ventrolateral medulla in SIDS cases, we hypothesized that infants without the full complement of neurons and neuropil (ARCn hypoplasia) are at risk for SIDS because they are unable to develop appropriate cardioventilatory control during this crucial developmental period.

Expression of apoptosis and proliferating cell nuclear antigen (PCNA) in the cardiac conduction system of crib death (SIDS).

Aim of this study is to determine the expression of apoptosis and Proliferating Cell Nuclear Antigen (PCNA) in the cardiac conduction system in crib death and explained death (ED) cases. Postnatal morphogenesis of the conducting tissue is an important part of its normal development. In the atrio-ventricular node (AVN) and His bundle (HB) it consists of degeneration, cell death and replacing in an orderly programmed way. However, its nature and its relation to crib death is not yet fully explained. Apoptosis and PCNA were investigated in 8 heart conduction systems of infants dying of crib death and in 3 conduction systems of infants dying of ED as controls. The cardiac conduction system was removed in two blocks: the first included the sino-atrial node (SAN) and the crista terminalis, the second contained the atrio-ventricular node (AVN), His bundle (HB), bifurcation, and bundle branches. In the conduction systems as well as in the common myocardium the PCNA Labeling Index (PCNA-LI) was found to be negative in all cases. The apoptotic indices (AI) in SIDS and in ED were found to have no statistically significant differences (p>0.05). The SAN, in both groups, showed an AI similar to the one detected in common myocardium. In almost all cases, TUNEL labeling was detected in peripheral region of the AVN, close to the atrial myocardium. The AI was higher in the AVN, HB and the initial tract of bundle branches than in the common myocardium (p<0.05; Student's t test).

The role of different biomarkers (DNA, PCNA, apoptosis and karyotype) in prognostic evaluation of superficial transitional cell bladder carcinoma.

BACKGROUND: In order to clarify the variable behaviour of transitional cell bladder carcinomas (TCBC) with same clinico-pathologic pattern, we investigated the prognostic significance of various biomarkers (PCNA, DNA, apoptosis, karyotype). MATERIALS AND METHODS: We studied 177 superficial TCBC (stage T1) undergoing transurethral resection (TUR). Analysis of biological indicators was performed on serial paraffin sections: DNA by static cytometry, karyotype by fluorescence in situ hybridisation (FISH), PCNA and apoptosis by immunohistochemistry. RESULTS: The most salient results are represented by prevalence of diploidy (56%), a mean PCNA labeling index (PCNA-LI) of 8.2%, nonrandom trisomies and tetrasomies of chromosome 7, and a high presence of apoptosis (in 72% of cases). These data were not related to histological grading. Multivariate analysis showed that only PCNA-LI is a an independent prognostic marker for patient survival (p = 0.01). Besides, we observed a worse prognosis in the presence of both very high PCNA indices and low or absent apoptosis. CONCLUSIONS: The results of this study suggest that tumor prognostic potential in TCBC should be evaluated on the basis of the association between PCNA cell kinetics and cell apoptosis information.

Chromosomal alterations in atherosclerotic plaques.

Alterations of chromosomes 7 and 11 have been involved in the progression of atherosclerosis. Twenty-three carotid endarterectomy specimens were studied for the presence of alterations in chromosomes 7 and 11, and fibroblastic growth factor-3 (FGF-3) gene amplification. Besides classic histological stainings, immunophenotyping of cellular and vascular components and fluorescence in situ hybridization (FISH) were performed. At the caps, unstable plaques (n=18) showed inflammatory infiltration of macrophages, smooth muscle cells, and T-lymphocytes. Specifically in these regions, the FISH showed varying percentages of trisomy (15/18) and tetrasomy (8/15) of chromosome 7. In four cases polisomy 7 was noted in some nuclei. Monosomy of chromosome 11 and gene amplification of FGF-3 gene was observed. The FISH of the five stable plaques and normal arterial walls showed no chromosome alterations; furthermore, chromosome 3, which is not involved in atherosclerotic progression, presented a normal ploidy of smooth muscle cells in stable and unstable plaques and normal arterial walls. In conclusion, chromosome 7 and 11 alterations and FGF-3 gene amplification are components of unstable plaques, and might contribute to the evolution of stable plaques into complicated plaques.

Inflammatory cells, apoptosis and Chlamydia pneumoniae infection in atherosclerosis.

Chlamydia pneumoniae (CP), chromosomal alterations and apoptosis were suggested as contributing factors in the pathogenesis of atherosclerosis. Early (EP) and unstable plaques (UP) were studied in order to assess infiltrate composition, the apoptotic index, chromosome 7 stability and to investigate the concurrent presence of CP in EP and UP. Paraffin embedded sections of three iliac arteries and four aortas from young donors (EP), and four coronaries and nine carotid arteries (UP) were used. Aside from histological techniques, immunophenotypification for macrophages, T and B cells, smooth muscle and endothelial cells; FISH and DNA nick end labeling were performed. The amplifications with PCR for CP infection were negative in all specimens. In the EP, a focal myointimal thickening with foam cells and scarce smooth muscle cells was observed. Macrophages were most frequent in the intima (10.8%) while T and B cells were found in 2.3 and 1.5%. In the UP a thin cap covering a lipid-rich core with widespread vascularization and with severe luminal obstruction was observed. Macrophages were increased (21%), and T (1.5%) and B cells (3.5%) in the caps and inner areas of the lipid cores. At these sites, the FISH showed trisomy and tetrasomy of chromosome 7 and apoptosis was very frequent (10-30%). Macrophages in intimal lesions is one of the most prominent, consistent and permanent features in EP, and an elevated apoptotic index and chromosome 7 instability might contribute to evolution from stable to complicated plaques, while CP seems to play no role. However, further studies are needed with more cases to confirm this last observation.

Sudden Infant Death Syndrome (SIDS): a study of cardiac conduction system.

The theory that Sudden Infant Death Syndrome (SIDS) may be related to lethal cardiac arrhythmias or heart block due to structural abnormalities of the conduction system is attractive and still of particular interest. We analyze 69 autopsied cases of SIDS (46 males and 23 females, infants ranging in age from 3 to 365 days) and 24 age-matched cases of explained death (ED) as controls (16 males and 8 females), infants who died from extracardiac cause (cerebral and respiratory). SIDS and ED groups were divided into three subgroups according to the age: (A) from 3 to 60 days; (B) from 61 to 120 days; (C) from 121 to 365 days. Histological observations were focused on the cardiac conduction system (CCS) which was examined on serial sections with the technique devised by one of the present authors (L. Rossi). The following findings were observed: resorptive degeneration (97.10% of SIDS, 75% of ED), His bundle dispersion (33. 33% of SIDS, 16.66% of ED), Mahaim fibers (21.73% of SIDS, 8.3% of ED), cartilaginous meta-hyperplasia (5.79% of SIDS, 4.16% of ED), persistent fetal dispersion (24.63% of SIDS, 16.66% of ED), intramural right bundle (20.29% of SIDS, 25% of ED), left sided His bundle (20.29% of SIDS and 8.3% of ED), hemorrhage of the atrio-ventricular junction (15.94% of SIDS), septation of the bifurcation (13.04% of SIDS), atrio-ventricular node (AVN) dispersion (7.24% of SIDS), sino-atrial node hypoplasia (5.79% of SIDS), Zahn node (1.45% of SIDS), His bundle hypoplasia (1.45% of SIDS), intramural left bundle (1.45% of SIDS), AVN dualism (2.89% of SIDS), and His bundle dualism (2.89% of SIDS, 4.16% of ED). Only the presence of resorptive degeneration was significantly higher in SIDS than in ED cases (p = 0.004). Regarding the subgroups, the only significant difference was the higher presence of intramural right bundle in SIDS-A than in SIDS-B (p = 0.01). Despite the non-specificity of most of these findings, we believe that these changes, associated with particular conditions and/or neurovegetative stimuli, could cause potentially malignant arrhythmias. These data suggest the need for an accurate approach and examination of the cardiac conduction system in all cases of sudden death in infancy

PCNA immunostaining as a valid alternative to tritiated thymidine-autoradiography to detect proliferative cell fraction in transitional cell bladder carcinomas.

BACKGROUND: The thymidine labeling index (T-LI) evaluated with the autoradiographic technique on fresh specimens is the most objective and specific marker of tumor proliferative cell fraction. In this study we investigated T-LI and PCNA expression in 68 cases of transitional cell bladder carcinomas in order to evaluate whether PCNA immunostaining in paraffin sections could be a valid alternative to autoradiographic methods for cell kinetic detection. Immunohistochemistry was applied after different fixatives: usual formalin, neutral 4% buffered formalin and Bouin. The results revealed that only in buffered formalin-fixed tumor specimens, considering the most darkly stained cells, the PCNA-LI in all cases overlapped with T-LI. Higher values than those observed with autoradiography were detected in PCNA values obtained after usual formalin fixation whereas no or faint PCNA staining was seen in material fixed in Bouin. Thus, only cells with strong PCNA staining can be considered as significant in evaluating the proliferative activity in transitional cell bladder carcinoma. By using immunohistochemical methods it should thus be possible to overcome many of the inherent difficulties of autoradiography and likewise to obtain accurate and significant information on the biological aggressiveness of bladder tumors and consequently of patient prognosis.

Severe hypoplasia of medullary arcuate nucleus: quantitative analysis in sudden infant death syndrome.

The human arcuate nucleus (ARCn) is postulated to be homologous to ventral medullary cells involved in chemoreception, and respiratory and blood pressure responses. Abnormalities in central respiratory control may result from dysfunction of this anatomic ventral area. We evaluated the changes of the neuronal population of the medullary ARCn in infants victims of the sudden infant death syndrome (SIDS). In this study we tested the hypothesis that anatomical deficiency of the ARCn is associated with SIDS. The volume and neuronal density of the ARCn were morphometrically quantified with an image analyzer in 36 cases of SIDS and 12 age-matched controls. We found a marked hypoplasia in the SIDS ARCn compared to controls and, particularly, in 11 SIDS cases (30%) in which the ARCn exhibited a severe hypoplasia, being almost totally absent. Three-dimensional reconstructions and morphometric measurements of ARCn confirmed this marked hypoplasia in all the serial sections examined (P = 0.0001) and the reduced neuronal density (P = 0.0025) in relation to control cases. In conclusion these abnormalities observed in the ARCn are consistent with the idea that ARCn dysfunction plays an important role among the causative factors of sudden infant death. The hypoplasia of the ARCn represents the most frequent congenital abnormality in our experience, and can be a plausible morphological substrate for a subset of SIDS.

[Proliferative activity and chromosomal alterations of smooth muscle cells in atherosclerosis]. / Actividad proliferativa y alteraciones cromosómicas de las células musculares lisas en la ateroesclerosis.

Atherosclerosis is the most frequent cause of death in industrialized countries. Lesions are characterized by lipid deposits, focal thickening of the arterial wall with proliferation of smooth muscle cells (SMC), mononuclear infiltrates and neoformed vessels. In this paper, we studied the proliferative characteristics and cytogenetic alterations of SMC. These cells, expressing specific muscular actin, were diploid with an increased proliferative index for PCNA. A high percentage of SMC showed intense expression of p53. There were signs of chromosomal instability, being the most frequent findings chromosome 7 trisomy and chromosome 11 monosomy. Additionally, the gene for FGF-3 showed a marked amplification. These findings strongly suggest that SMC proliferation is active, and is related to the accumulation or mutation of the p53 oncoprotein. It also presents specific chromosomal alterations in close relation with growth factors. According to these findings SMC hyperplasia in the atherosclerosis plaque may be considered as a cellular clonal expansion.

Actividad proliferativa y alteraciones cromosómicas de las células musculares lisas en la ateroesclerosis. / [Proliferative activity and chromosomal alterations of smooth muscle cells in atherosclerosis]

Atherosclerosis is the most frequent cause of death in industrialized countries. Lesions are characterized by lipid deposits, focal thickening of the arterial wall with proliferation of smooth muscle cells (SMC), mononuclear infiltrates and neoformed vessels. In this paper, we studied the proliferative characteristics and cytogenetic alterations of SMC. These cells, expressing specific muscular actin, were diploid with an increased proliferative index for PCNA. A high percentage of SMC showed intense expression of p53. There were signs of chromosomal instability, being the most frequent findings chromosome 7 trisomy and chromosome 11 monosomy. Additionally, the gene for FGF-3 showed a marked amplification. These findings strongly suggest that SMC proliferation is active, and is related to the accumulation or mutation of the p53 oncoprotein. It also presents specific chromosomal alterations in close relation with growth factors. According to these findings SMC hyperplasia in the atherosclerosis plaque may be considered as a cellular clonal expansion.

Evidence for apoptosis in non-small cell lung carcinoma. Relationship with cell kinetics and prognosis.

OBJECTIVE: To investigate cell death via apoptosis in non-small cell lung carcinoma (NSCLC) and its correlation with proliferative indices and follow-up. STUDY DESIGN: In 38 cases of NSCLC (21 squamous cell carcinomas and 17 adenocarcinomas) we analyzed apoptosis by nuclear morphology and in situ DNA fragmentation end labeling and the cell kinetics by an autoradiographic method with tritiated thymidine and by immunohistochemistry with anti-proliferating cell nuclear antigen (anti-PCNA) antibodies. We also evaluated mitotic frequency. Apoptotic index (AI) was correlated with the thymidine and PCNA labeling indices (T-LI and PCNA-LI, respectively) and with the mitotic index. RESULTS: The percentage of proliferating cells (T-LI range, 0.1-20.1%; PCNA-LI range, 0-14.7%) was generally considerably higher than that of apoptotic cells (range, 0-8%) and of mitotic cells (range, 0.1-1%). Survival at six years was significantly higher in patients with high levels of apoptosis and low T-LI values. CONCLUSION: AI and T-LI are independent and very useful prognostic factors in NSCLC. A high percentage of proliferating cells in terms of T-LI correlates with poor prognosis, whereas a high AI indicates a favorable outcome.