TNF-alpha inhibits insulin action in liver and adipose tissue: A model of metabolic syndrome.

Several studies suggest that TNF-alpha contributes to the development of insulin resistance (IR). We compared transcriptional profiles of rat H-411E liver cells exposed to insulin in the absence or presence of TNF-alpha. We identified 33 genes whose expression was altered by insulin, and then reversed by TNF-alpha. Twenty-six of these 33 genes created a single network centered around: insulin, TNF-alpha, p38-MAPK, TGFb1; SMAD and STAT1; and enzymes and cytokines involved in apoptosis (CASP3, GADD45B, IL2, TNF-alpha, etc.). We analyzed our data together with other data of gene expression in adipocytes and found a number of processes common to both, for example, cell death and inflammation; intercellular signaling and metabolism; G-Protein, IL-10 and PTEN signaling. Moreover, the two datasets combined generated a single molecular network that further identified PTEN (a phosphatase) as a unique new link between insulin signaling, IR, and apoptosis reflecting the pathophysiology of "metabolic syndrome".

Characterization of early developments in the splenic leukocyte transcriptome of NOD mice.

Transcriptome analysis is a powerful tool to characterize changes in leukocyte gene expression patterns and reveal very early molecular abnormalities in tissue. Herein, we report on characterization of the very earliest abnormalities in the transcriptome of leukocytes from young "prepathologic" NOD and NON female mice.

Forensic pathological aspects of postmortem imaging of gunshot injury to the head: documentation and biometric data.

To determine the value of imaging procedures such as computer tomography (CT) and magnetic resonance imaging (MRI) of the head in providing additional information of forensic relevance, we examined 17 cadavers of human victims of gunshot wounds to the head. Three of the victims briefly survived the gunshot wound. The weapons involved were all guns with low muzzle energy (<550 J), i.e., handguns and low-velocity rifles. In the majority of cases ( n=15) a penetrating wound to the head was found, only two cases showed the bullet lodged in the brain. In some cases, imaging of the skull and brain was performed prior to autopsy; in others imaging took place after autopsy on the isolated, formalin-fixed brain. The imaging findings were correlated with the criminological data and the results of macroscopic and microscopic examination of the brain. The findings on the bony structures of the head provided imaging criteria for differentiation between entrance and exit of the gunshot wounds, which corresponded to the forensic pathological findings at autopsy. CT scans and MRI of the cerebral parenchyma revealed lanes of opaque bone and missile fragments along the course of the missile, which allowed recognition of the missile track in 3D reconstruction. Biometric reconstruction allowed easy determination of the angle of the missile track in all three planes. Examination of the parenchymal structures and imaging of the isolated, formalin-fixed brain enabled tracking of the missile path directly along the zone of destruction as well as demonstration of secondary changes such as air bubbles along the bullet course, hemorrhage and edema. The significance of a translucent zone surrounding the missile track in several cases remains unclear; it probably represents tissue destruction secondary to temporary cavitation. The imaging procedures described here allowed excellent documentation of in situ conditions, while the storing of data enabled biometric reconstruction for determination of the angle of trajectory, of entrance and exit wounds, and the extent of tissue damage along the missile track and, possibly, in the zone of temporary cavitation.

Improved in vivo pancreatic islet function after prolonged in vitro islet culture.

BACKGROUND: Difficulties with recovering and preserving pancreatic islets have hampered progress in islet transplantation. In previous in vitro studies, our laboratory successfully demonstrated that using serum-free medium for prolonged pancreatic islet culture allows postculture recovery ratios greater than those obtained with standard media with sustained in vitro islet function. The goal of this study was to determine whether culturing of islets in a modified serum-free medium (M-SFM) would sustain function in vivo. METHODS: Islets were isolated from pancreata procured from 12 cadaveric organ donors and cultured in the M-SFM for up to 2 months, cryopreserved at -70 degrees C within 1-3 days of isolation for 2 months, or placed in short-term culture (3-5 days) before their transplantation under the kidney capsule of nonobese diabetic-severe combined immunodeficient mice (n=4-7 per group/time point). In vivo islet function was assessed by measuring the production of human insulin and C-peptide over a period of 3-15 months. RESULTS: After extended culture of islets in M-SFM for 1 or 2 months, transplanted islets maintained their viability, and in some instances in vivo function improved when compared with short-term cultured islets transplanted from the same preparation (P<0.01). Improvement was particularly evident for islets cultured for 1 month. Furthermore, when compared with cryopreserved preparations, early function (postoperative day 7) of islets from 1-month culture preparations was statistically better (P<0.05). Prolonged culture in M-SFM had no significant impact on long-term function, inasmuch as cultured islets functioned for more than 120 days. CONCLUSION: These data demonstrate that prolonged islet culture in M-SFM sustained viability and function, and in some instances had a positive effect on in vivo islet function, particularly in the 1-month cultures. No negative effect on long-term in vivo function was demonstrated in this study. Confirmation in clinical models utilizing extended (1-2 months) islet culture in M-SFM could significantly enhance islet transplantation by allowing the identification of best-matched recipients, pretransplantation recipient conditioning, and possible pretransplantation islet modifications to promote engraftment and prolonged graft function.

Creutzfeldt-Jakob disease (CJD) in a case of suspected chronic heavy metal poisoning.

We describe a patient who died of suspected heavy metal poisoning after a nine-month history of rapidly worsening dementia. Autopsy at a forensic-pathological institute established the postmortem diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) based on demonstration of the proteinase-resistant prion protein (PrPsSc) in Western-Blot on native brain tissue. Microscopic examination of the macroscopically largely inconspicuous brain revealed marked spongiform changes in the gray matter--mainly affecting the cerebral cortex, nucleus caudatus, and putamen--with confluent vacuoles. Patchy or perivacuolar deposits of PrPSc were found as well as granular PrPsc deposits. The cerebellum contained focal PrPsc deposits. There was an astrogliosis in the white matter and a proliferation of microglia in the gray matter with a simultaneous clear reduction in neuronal elements. The differential diagnosis is discussed, as is the potential risk to those performing autopsy on forensic cases with a clinical picture of rapidly progressing dementia, especially in cases where a prion disease is not initially suspected.

Death from thermal effects and burns.

One hundred and fifteen unselected autopsy cases of death from thermal effects and/or fire between 1990 and 1999 were analyzed with regard to time of death, signs of vitality at the scene of the fire, the manner and cause of death, and the extent of soft tissue loss. The cases represented approximately 6% of all autopsy cases at the Institute of Legal Medicine responsible for a catchment area with approximately 700,000 inhabitants. In 23 victims suffering burn injuries, death occurred during initial medical care or clinical treatment. The causes of death were primary respiratory arrest due to smoke poisoning or delayed shock caused by thermal injuries to the skin. Death occurred at the scene of the fatal event in 85 cases: eight cases exhibited no thermal effects; the cause of death in one of these cases was polytrauma incurred in a leap from a height; in seven cases there was poisoning due to smoke inhalation. The remaining 77 cases were characterized by signs of intensive thermal and/or fire effects. Clear signs of vitality (carboxyhemoglobin (COHb) in blood, inhalation and/or swallowing of soot) were found in 84.7% of the victims dying at the site of the fatal event. Of the 13 victims showing no signs of vitality at the scene, a cause of death could be determined in only seven cases; death in the other six cases remains unexplained. Quantification of the soft tissue loss revealed a possible correlation with the temperature and time course of heat exposure.

Petechiae of the baby's skin as differentiation symptom of infanticide versus SIDS.

The successive killing of three siblings by their biological mother at two-year intervals is described. The children were 367 days, 75 days and 3 years old. Although sudden infant death syndrome (SIDS) or interstitial pneumonia could not be ruled out as the cause of death in the two younger children, who were killed first, the third child exhibited discrete signs of violence in the mouth and throat area which were interpreted as proof of infanticide. All three children had petechiae of the skin of the face and throat, the upper thorax, the shoulders and the mucous membranes of the mouth. None of the children exhibited signs of a disease-related hemorrhagic tendency. After the mother was convicted of murdering the three-year-old boy by smothering in combination with compression of the thorax, she confessed to having killed the other two children in a similar manner. In the absence of hemostatic disease, the presence of petechiae of the skin extending over the entire drainage area of the Vena cava superior can be regarded as evidence of an increase in pressure in the thoracic cavity secondary to obstruction of the airways with simultaneous chest compression.

Sudden unexpected infant death due to fibroma of the heart.

A 7-month-old previously healthy female infant was found dead in her crib by her mother shortly after having been laid down to sleep following the noontime feeding. Because the child did not suffer from an acute illness and no other evidence pointed to a cause of death, it was initially assumed by the police that she had died of sudden infant death syndrome. At autopsy, however, the cause of death was determined to be cardiac arrhythmia secondary to fibroma of the heart.

Human pancreatic islets transfected to produce an inhibitor of TNF are protected against destruction by human leukocytes.

The objective of this study was to determine whether transfection of human islets with an adenovirus construct encoding an inhibitor of tumor necrosis factor (TNFi) was effective at limiting damage to beta cells induced by human peripheral blood leukocytes (huPBL). Human islets transfected with TNFi or control islets were transplanted under the kidney capsule of NOD-scid mice. After a 15-day engraftment period, half of the mice received injections of activated huPBL and half received buffer injections. Islet graft function was assessed by two different methods, both of which use a species-specific radioimmunoassay to determine human insulin. In some mice, insulin production following intraperitoneal glucose injection was determined in serum. In other mice, total graft insulin content was determined by acid ethanol extraction. Histochemical stains were performed on some kidneys at the termination of the experiment to evaluate graft presence, transgene expression, and huPBL infiltration. In huPBL injected mice, graft performance was maintained in mice whose grafts were transfected with TNFi but declined substantially in control groups with sham transfected or beta-galactosidase transfected islet grafts. Similar results were obtained using either glucose-stimulated insulin release or graft insulin content as a measure of graft survival. There was no significant difference in graft function between control groups receiving buffer injections, regardless of whether the islets had been transfected. Human leukocytes were found in all huPBL groups regardless of islet transfection status. We conclude that transfection of human islets with an adenovirus encoding TNFi protects beta cells from destruction induced by human leukocytes. The local production of TNFi does not prevent graft infiltration by leukocytes, only the destruction of grafts by the infiltrating leukocytes. These results raise the possibility that local expression of an inhibitor of the proinflammatory cytokine TNF-alpha may also prevent graft failure in clinical islet transplantation.

[Fatal child neglect in West Germany 1 January 1985 to 2 October 1990. Results of a multicenter study]. / Tödliche Kindesvernachlässigung in der Bundesrepublik Deutschland im Zeitraum 1.1.1985 bis 2.10.1990. Ergebnisse einer multizentrischen Studie.

UNLABELLED: Up to now reliable data were available on cases of lethal child neglect in the area of the Federal Republic of Germany prior to reunification (the former West Germany). In a multicenter study we therefore examined the police and court records for such cases occurring in the period from 1 January 1985 to 2 October 1990 in nearly the entire area of Federal Republic of Germany. RESULTS: The study center received information on 19 cases of lethal child neglect. Extrapolated to all institutes of legal medicine, this corresponds to 20 cases and thus 3.5 cases a year in the whole of West Germany in the period studied. There is to be added a dark-field which cannot be limited more precisely. However, the cases of fatal child neglect might have occurred much more seldom than fatal child abuse caused by use of physical violence. Slightly more than half the victims were younger than 1 year, the oldest one was 7 10/12 years old. Most frequently the children died of starvation and thirst. Mostly the mothers/nursing mothers killed the child alone or together with the victim's father/stepfather. In the majority of the cases there was not a close affection between parents and child. Nearly 30% female/male perpetrators suffered from chronic alcohol abuse. Only 15 (= 56%) of 27 female/male perpetrators were sentenced to imprisonment (period between 7 months on probation and 10 years). Mitigation circumstances existed for nearly half the persons sentenced to imprisonment. It is true that child neglect is a rarer crime, but the experts of legal medicine always have to indicate errors made during the external inspection of the corpse (among others failures to see indications of neglect).

[Fatal child abuse (caused by physical violence) in Germany during 1 January 1985 to 2 October 1990. Results of a multicenter study]. / Tödliche Kindesmisshandlung (durch physische Gewalteinwirkung) in der Bundesrepublik Deutschland im Zeitraum 1.1.1985 bis 2.10.1990. Ergebnisse einer multizentrischen Studie.

UNLABELLED: No reliable data are available on cases of lethal child abuse (by active force) in the area of Federal Republic of Germany prior to reunification (the former West Germany). In a multicenter study we therefore examined the police and court records for such cases occurring in the period 1 January 1985 to 2 October 1990 in nearly the entire area of Federal Republic of Germany. RESULTS: The study center received information on 58 cases of lethal child abuse. Extrapolated to all institutes of legal medicine, this corresponds to 62 cases in all of West Germany in the period studied. An approximately equal number of unreported cases should be added to this figure. Including unreported cases, at least 20 cases of lethal child abuse occurred per year; thus only one in every two cases ever came to light. Almost two thirds of the victims were younger than one year old. At autopsy 59% exhibited signs of repeated abuse at autopsy. By far the most common cause of death was direct impact from a blunt object, usually to the head. Mostly, the male person to whom the victim relates most closely (father, stepfather, partner of the mother) has killed the child. Twenty-one of the 74 persons charged saw the charges against them dropped or were acquitted due to lack of evidence; 51 received sentences ranging from one year probation to life. In the remaining two cases the outcome of the trial was unknown. Signs of abuse were readily apparent at autopsy in almost all cases. The high number of unreported cases underscores the need to educate medical students and practicing physicians to be on the look-out for signs of abuse and argues for an increase in the rate of autopsy.

Retardation or acceleration of diabetes in NOD/Lt mice mediated by intrathymic administration of candidate beta-cell antigens.

A single injection of syngeneic islet cells into the thymus of 4-week-old NOD/Lt female mice strongly retards diabetogenesis. The present study used the intrathymic route of antigen administration to compare the relative efficacy of peptides/proteins derived from two major candidate pancreatic beta-cell autoantigens, insulin and GAD65, to modulate diabetogenesis. Intrathymic administration of insulin B chain or recombinant human GAD65 significantly suppressed diabetogenesis during a 20-week follow-up period, whereas no protection was mediated by either insulin A chain or a synthetic peptide (A2) derived from it. Quite unexpectedly, two GAD65-derived peptides near the COOH-terminus (p34 and p35) accelerated diabetes onset. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed on cDNAs from isolated islets or whole pancreases of NOD/Lt females 4 weeks after intrathymic injections. Protection mediated by intrathymic administration with either intact islet cells or GAD65 were correlated with an upregulation of mRNA for T-helper 2 (Th2)-associated cytokines (interleukin [IL]-4, IL-10), concomitant with downregulation of Th1-associated interferon (IFN) transcripts (all normalized to T-cell receptor Cbeta transcripts) in islet-infiltrating lymphocytes. Protection mediated by the intrathymic administration of insulin B chain, however, correlated only with a modest upregulation of IL-4 and IL-10 transcript levels, and no diminution in IFN-gamma transcripts. In contrast, the diabetes-accelerating GAD65 p34 and p35 peptides were not associated with an immune deviation, expressing levels of IFN-gamma characteristic of islet-infiltrating lymphocytes in vehicle-injected NOD controls. Hence, Th1-to-Th2 immune deviation provides only a partial explanation for peptide immunotherapy of diabetes in NOD mice. The finding that certain peptides can accelerate rather than retard diabetogenesis as a function of route and age of administration adds a cautionary note to this type of therapy.

Initiation of autoimmune diabetes in NOD/Lt mice is MHC class I-dependent.

MHC class II alleles clearly contribute a primary genetic component of susceptibility to autoimmune insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. However, IDDM does not occur in NOD mice made MHC class I-deficient by a functionally inactivated beta2-microglobulin allele (beta2m(null)). In the present study the beta2m(null) mutation was used to examine the relative contributions of MHC class I and class II-dependent T cell responses for initiating autoimmune pancreatic beta cell destruction in NOD mice. Splenocytes from diabetic NOD donors transferred IDDM to both lymphocyte-deficient NOD-scid (class I+) and NOD-scid.beta2m(null) mice (class I-). In contrast, splenocytes from young prediabetic NOD donors only transferred IDDM to class I+, but not class I- NOD-scid recipients. However, splenocytes from prediabetic NOD donors did transfer IDDM to NOD-scid.beta2m(null) recipients previously engrafted with class I+, but not class I-, pancreatic islets. CD4+ T cell lines reactive against some syngeneic class I+ targets could be isolated from NOD.beta2m(null) mice. However, NOD.beta2m(null) T cells underwent activation-driven deletion when transferred into class I+ NOD-scid recipients. Hence, the class I autoreactive T cells present in NOD.beta2m(null) donors did not elicit IDDM when transferred into class I+ NOD-scid recipients. Collectively, these results show that autoimmune IDDM in NOD mice is initiated by MHC class I-dependent T cell responses, but this leads to the subsequent activation of additional T cell populations that can mediate pancreatic beta cell destruction in a MHC class I-independent manner.

The role of enteroviral infections in the development of IDDM: limitations of current approaches.

Enteroviruses have been examined for their possible role in the etiology of IDDM for nearly 40 years, yet the evidence remains inconclusive. The mechanism of acute cytolytic infection of beta-cells, proposed by earlier studies, appears to be incompatible with the long preclinical period of autoimmunity preceding IDDM. Advances in molecular biology have improved our understanding of enteroviral biology and of potential alternative pathogenic mechanisms through which enteroviruses may cause diabetes. The focus of future human studies will likely shift from people with IDDM to those with prediabetic autoimmunity to determine whether acute enteroviral infections can promote progression from autoimmunity to overt diabetes. We propose that such studies use assays to detect enteroviral RNA, in addition to IgM serology. RNA assays can overcome sensitivity and type-specificity limitations of IgM assays as well as identify diabetogenic strains of enteroviruses, if such exist. Evaluation of the role of enteroviruses in triggering beta-cell autoimmunity in humans will require large prospective studies of young children. The Diabetes Autoimmunity Study in the Young--one of very few such studies currently underway--is focusing on potential interactions between HLA class II genes and enteroviral infections. Future studies will likely examine interactions between viral infections and non-HLA IDDM candidate genes, including those that may determine beta-cell tropism of candidate viruses.

[Sudden fatalities in mechanically restrained patients]. / Plötzliche Todesfälle mechanisch fixierter Patienten.

We report on 7 nursing home or hospital patients who died suddenly and unexpectedly during physical restraint. Four of the patients were found dead hanging beside their beds, with their waist restraints displaced to the thorax. In spite of a variety of preexisting diseases, asphyxia by thorax compression was the most probable cause of death. Three other patients, when falling out of their beds, were strangulated by the head opening of a nursing bedcover fixed to the bed. In all instances the fatal accidents resulted from improper handling of the restraint devices, namely from the omission of bed rails as well as of the obligatory waist belt lateral fixations. The bedcover type involved in three fatalities is destined for care purposes but not licensed as a restraint device. Physical restraint fatalities can be avoided to a large extent if the producers' instructions are strictly observed, and only especially trained and supervised personnel is admitted to this field of duties.

The thymus as a site for evaluating the potency of candidate beta cell autoantigens in NOD mice.

Intrathymic (i.t.) injection of islet cells or whole islets retards development of insulin dependent diabetes mellitus (IDDM) in spontaneous animal models of the disease. Protection of 4-week-old prediabetic NOD/Lt female mice from subsequent IDDM development was specific for the it route of administration since intraperitoneal injection of an equal number of syngeneic islets failed to retard IDDM. The protective effect of i.t. injection of islet cells was compared with the effect of i.t. injection of syngeneic peritoneal exudate cells, NIT-1 cells, bovine serum albumin (BSA), ABBOS peptide, a 52 kDa islet cell membrane protein, various synthetic peptides from human glutamic acid decarboxylase (GAD) and a Coxsackievirus B4-derived peptide with homology to GAD. Interestingly, only a GAD-derived peptide containing sequence homology to Coxsackie-virus B4, and the corresponding Coxsackievirus B4-derived peptide, delayed IDDM onset. To establish the immunological mechanism underlying the reduced IDDM incidence following i.t. injection of islet cells, adoptive transfer of splenic leukocytes into NOD-scid/scid mice was performed. Splenic leukocytes from i.t.-injected non-diabetic females transferred IDDM into NOD-scid/scid recipients, but more slowly than splenocytes from unmanipulated, diabetic (control) donors. Co-transfer of 1:1 mixtures of splenic leukocytes from it islet-injected (and diabetes-free) NOD/Lt females and from untreated NOD/Lt diabetic donors produced IDDM as rapidly as splenocytes from diabetic donors injected alone. Hence, any peripheral suppression generated in i.t.-protected females was not sufficiently strong to prevent IDDM transfer by committed T-effector cells from the diabetic donors.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple low-dose streptozocin-induced diabetes in NOD-scid/scid mice in the absence of functional lymphocytes.

The murine severe combined immunodeficiency (scid) mutation was used to assess whether the diabetogenic effects of multiple low-dose streptozocin (MD-STZ) administration required the presence of functional T-cells. An STZ dose as low as 30 mg/kg body wt for 5 days induced hyperglycemia in young NOD/Lt-+/+ male mice, whereas a dose of 50 mg/kg for 5 days was required to elicit comparable hyperglycemia in C.B.-17-+/+ male mice. The greater NOD strain sensitivity was not a function of preexisting insulitis, because insulitis- and diabetes-free NOD male mice congenic for a diabetes-resistant major histocompatibility complex haplotype were equally susceptible to MD-STZ. This was confirmed in NOD-scid/scid and C.B.-17-scid/scid males. Both were completely insulitis-free, and despite the absence of functional T- cells and B-cells, both congenic stocks were as sensitive to MD-STZ as congenic +/+ controls. Indeed, MD-STZ-induced hyperglycemia in NOD-scid/scid male mice was significantly higher than in NOD/Lt-+/+ male mice. The NOD-scid/scid mouse as a recipient of adoptively transferred splenocytes clearly delineated a distinct pathogenesis of spontaneous insulin-dependent diabetes mellitus (IDDM) versus MD-STZ-induced hyperglycemia. Splenocytes from spontaneously diabetic NOD/Lt males, but not those from donors given MD-STZ, readily transferred IDDM, even when host beta-cells were sensitized by a single injection of STZ before adoptive transfer. We conclude that IDDM induced by MD-STZ is not mediated by T-cell- or B-cell-dependent autoimmune mechanisms in a fashion analogous to the spontaneous IDDM characteristic of NOD mice.

Myocardial fibrosis--a rare complication in patients with cystic fibrosis.

We report a 10-month-old male infant who was admitted to our hospital with a history of failure to thrive and bulky stools. On examination, he was dystrophic and had a protruding abdomen, but he was well oxygenated and his lungs were clear on auscultation. A tachycardia of 145 beats per min and radiological evidence of cardiomegaly indicated involvement of the heart, but an ECG failed to show signs of myocarditis or cardiac hypertrophy. An elevated sweat chloride concentration of 141 mEq/l confirmed the diagnosis of cystic fibrosis (CF). Molecular analysis revealed heterozygosity for the common mutation delta F508. He died unexpectedly of a sudden cardiac arrest 2 days later. Autopsy revealed scattered myocardial necrosis and fibrosis. Some 50 documented cases of myocardial fibrosis in infants with CF have been reported. Suggested causes such as malnourishment and hypovitaminosis remain speculative as systematic studies have yet to be done.