Generating Human iPSC-Derived Astrocytes with Chemically Defined Medium for In Vitro Disease Modeling.

To better understand and model neurological, in particular neurodegenerative diseases, human induced pluripotent stem cells (hiPSCs) offer a great source for generation of neural cells. We provide a protocol for the differentiation of hiPSc-derived astrocytes in vitro. This protocol not only is chemically defined, that is, it does not use serum, but also allows for the expansion of astrocyte progenitor cells and mature astrocytes. Large batches of hiPSc-derived astrocytes can be stored and used for defined in vitro disease models.

Wounding potential of 4.4-mm (.173) caliber steel ball projectiles.

From time to time, severe or fatal injuries caused by small caliber air rifle projectiles are seen. In forensic sciences, the theoretical wounding potential of these weapons and projectiles is widely known. Usually, shots against the skull were reported and, in these cases, penetrating the eyes or thin bone layers of the temporal region. Amongst a huge number of different projectiles available for air guns, sub-caliber 4.4-mm (.173) caliber steel ball projectiles were used in an unusual suicide case. This case led to fundamental questions concerning wound ballistics. An 82-year-old man shot once against his right temporal region and twice into his mouth with a 4.5-mm (.177) caliber air rifle. Because of the exceptionally deep penetration of the base of the skull and the use of spherical-shaped sub-caliber air rifle projectiles, terminal ballistic features were analyzed and compared to results published in forensic literature. Test shots using the same weapon and similar projectiles were fired into ballistic gelatin to measure and calculate basic wound ballistic variables of cal. 4.4-mm (.173) steel balls. In comparison, further test shots with cal. 4.5-mm (.177) steel balls BB (ball bearing), flat-headed and pointed air rifle pellets ("diabolos") were carried out. The theoretical penetration depth in solid bone was calculated with 36.4 mm, and test shots in gelatin from hard contact produced an on-average wound track of 120 mm underlining the potential wounding effect. Furthermore, spherical projectiles could roll back and forth within the barrel, and an air cushion between projectile and breechblock can reduce muzzle velocity by more than half, explaining the retained missile in the temporal region.

Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

Relevance of medical reports in criminal investigations of cases of suspected child abuse.

If a case of physical child abuse is suspected in Germany, the general feeling is often that "it does not matter whether you make a report or not" because, generally, no conviction is made anyway. This study investigates the juridical analysis of complaint cases of physical child abuse [criminal complaint parag. 225 StGB (German penal code) with filial victim]. It focuses on the doctor's role and the impact of their practice in relation to a later conviction. It is based on the analysis of 302 files of the enquiry from 2004-2009 from the department of public prosecution in Cologne, Germany. Besides general epidemiological data on the reporting person, the affected child and the presumed offender, the documents were reassessed for the relevance of medical reports for successful convictions. Only 7% (n = 21) of 302 complaints led to a conviction. In 38.1% (n = 8) of those cases, a medical report was mentioned as a piece of evidence, and just in two cases a (legal) medical report was quoted and mentioned as relevant for the conviction. 50% of the complaint cases with legal medical expertise led to a trial. In contrast, only 30.2% with a common medical report and 7.3% without a report led to a trial. The results show how a medical report existed in only a few cases. In those cases, the rate of performed trials was higher than for those without a medical report, but the report played a minor part when reasoning a verdict.

Absence of CCL2 and CCL3 Ameliorates Central Nervous System Grey Matter But Not White Matter Demyelination in the Presence of an Intact Blood-Brain Barrier.

A broad spectrum of diseases is characterized by myelin abnormalities, oligodendrocyte pathology, and concomitant glia activation, among multiple sclerosis (MS). Our knowledge regarding the factors triggering gliosis and demyelination is scanty. Chemokines are pivotal for microglia and astrocyte activation and orchestrate critical steps during the formation of central nervous system (CNS) demyelinating lesions. Redundant functions of chemokines complicate, however, the study of their functional relevance. We used the cuprizone model to study redundant functions of two chemokines, CCL2/MCP1 and CCL3/MIP1α, which are critically involved in the pathological process of cuprizone-induced demyelination. First, we generated a mutant mouse strain lacking functional genes of both chemokines and demonstrated that double-mutant animals are viable, fertile, and do not present with gross abnormalities. Astrocytes and peritoneal macrophages, cultured form tissues of these animals did neither express CCL2 nor CCL3. Exposure to cuprizone resulted in increased CCL2 and CCL3 brain levels in wild-type but not mutant animals. Cuprizone-induced demyelination, oligodendrocyte loss, and astrogliosis were significantly ameliorated in the cortex but not corpus callosum of chemokine-deficient animals. In summary, we provide a novel powerful model to study the redundant function of two important chemokines. Our study reveals that chemokine function in the CNS redounds to region-specific pathophysiological events.

Maternal serum copeptin, MR-proANP and procalcitonin levels at 11-13 weeks gestation in the prediction of preeclampsia.

PURPOSE: We investigated the potential value of maternal serum copeptin, midregional proatrial natriuretic peptide (MR-proANP) and Procalcitonin (PCT) levels at 11-13 weeks' gestation in the prediction of preeclampsia (PE) in a case-control study. MATERIALS AND METHODS: Maternal serum concentration of copeptin, MR-proANP and PCT were measured at 11-13 weeks' gestation in cases of PE (n = 35) and controls (n = 100). The PE group was divided into early-onset PE (EO-PE) and late-onset PE (LO-PE). From the regression model, the value in each case and control was expressed as a multiple of the expected median (MoM). The Mann-Whitney test was used to determine the significance of differences in the median MoM in each outcome group from that in the controls. RESULTS: In the PE group, compared to controls, maternal serum concentrations of copeptin, MR-proANP and PCT were not significantly different. CONCLUSION: The maternal serum copeptin, MR-proANP and PCT levels are higher in EO-PE and LO-PE patients, but the difference is not significant. Thus, their levels in first trimester are not proven to be effective markers to screen for PE.

CXCL10 triggers early microglial activation in the cuprizone model.

A broad spectrum of diseases is characterized by myelin abnormalities and/or oligodendrocyte pathology. In most, if not all, of these diseases, early activation of microglia occurs. Our knowledge regarding the factors triggering early microglia activation is, however, incomplete. In this study, we used the cuprizone model to investigate the temporal and causal relationship of oligodendrocyte apoptosis and early microglia activation. Genome-wide gene expression studies revealed the induction of distinct chemokines, among them Cxcl10, Ccl2, and Ccl3 in cuprizone-mediated oligodendrocyte apoptosis. Early microglia activation was unchanged in CCL2- and CCL3-deficient knockouts, but was significantly reduced in CXCL10-deficient mice, resulting in an amelioration of cuprizone toxicity at later time points. Subsequent in vitro experiments revealed that recombinant CXCL10 induced migration and a proinflammatory phenotype in cultured microglia, without affecting their phagocytic activity or proliferation. In situ hybridization analyses suggest that Cxcl10 mRNA is mainly expressed by astrocytes, but also oligodendrocytes, in short-term cuprizone-exposed mice. Our results show that CXCL10 actively participates in the initiation of microglial activation. These findings have implications for the role of CXCL10 as an important mediator during the initiation of neuroinflammatory processes associated with oligodendrocyte pathology.

Rate of deaths due to child abuse and neglect in children 0-3 years of age in Germany.

In recent years, increasing attention has been paid to the issue of (fatal) child abuse and neglect, largely due to the media attention garnered by some headline-grabbing cases. If media statements are to be believed, such cases may be an increasing phenomenon. With these published accounts in mind, publicly available statistics should be analysed with respect to the question of whether reliable statements can be formulated based on these figures. It is hypothesised that certain data, e.g., the Innocenti report published by UNICEF in 2003, may be based on unreliable data sources. For this reason, the generation of such data, and the reliability of the data itself, should also be discussed. Our focus was on publicly available German mortality and police crime statistics (Polizeiliche Kriminalstatistik). These data were classified with respect to child age, data origin, and cause of death (murder, culpable homicide, etc.). In our opinion, the available data could not be considered in formulating reliable scientific statements about fatal child abuse and neglect, given the lack of detail and the flawed nature of the basic data. Increasing the number of autopsies of children 0-3 years of age should be considered as a means to ensure the capture of valid, practical, and reliable data. This could bring about some enlightenment and assist in the development of preemptive strategies to decrease the incidence of (fatal) child abuse and neglect.

Short-term cuprizone feeding verifies N-acetylaspartate quantification as a marker of neurodegeneration.

Proton magnetic resonance spectroscopy (1H-MRS) is a quantitative MR imaging technique often used to complement conventional MR imaging with specific metabolic information. A key metabolite is the amino acid derivative N-Acetylaspartate (NAA) which is an accepted marker to measure the extent of neurodegeneration in multiple sclerosis (MS) patients. NAA is catabolized by the enzyme aspartoacylase (ASPA) which is predominantly expressed in oligodendrocytes. Since the formation of MS lesions is paralleled by oligodendrocyte loss, NAA might accumulate in the brain, and therefore, the extent of neurodegeneration might be underestimated. In the present study, we used the well-characterized cuprizone model. There, the loss of oligodendrocytes is paralleled by a reduction in ASPA expression and activity as demonstrated by genome-wide gene expression analysis and enzymatic activity assays. Notably, brain levels of NAA were not increased as determined by gas chromatography-mass spectrometry and 1H-MRS. These important findings underpin the reliability of NAA quantification as a valid marker for the paraclinical determination of the extent of neurodegeneration, even under conditions of oligodendrocyte loss in which impaired metabolization of NAA is expected. Future studies have to reveal whether other enzymes are able to metabolize NAA or whether an excess of NAA is cleared by other mechanisms rather than enzymatic metabolism.

Fatal postpartum air embolism due to uterine inversion and atonic hemorrhage.

We report a case of a 19-year-old woman who developed a persistent uterine hemorrhage after spontaneous delivery of a healthy child. Emergency laparotomy was indicated and then begun under stable circulatory conditions. Cardiac arrest occurred during the course of massive manual compression and packing of the uterus. After successful resuscitation, a supracervical hysterectomy was performed. During the suturing of the remaining cervix, a second cardiac arrest followed. The procedure was completed under constant external heart massage. Resuscitation was terminated due to the persistence of widened pupils. An autopsy was ordered by the public prosecutor as the manner of death was declared to be unascertained. An X-ray and a CT scan prior to the autopsy showed extensive gas embolism in both arterial and venous vessels extending from the pelvic region to the head. During the autopsy, gas was collected by aspirometer from the right ventricle of the heart. The autopsy showed no additional relevant findings, and gas analysis confirmed the suspicion of air embolism. The histological examination of the excised uterus especially in the corpus/fundus revealed an edema of the local smooth muscle cells and dilated vessels showing no sign of thrombogenesis. Upon evaluation of the clinical records, it became evident that, in addition to uterine atony, there had been a complete uterine inversion. This inversion was manually repositioned. After this maneuver, manual compression was performed. The air embolism, thus, was a complication of the manual repositioning of the uterine inversion. There is no evidence for other possible entries of the detected gas. In order to perform an effective exploration, the availability of all clinical records should be mandatory for medico-legal investigations of unexpected postpartum deaths.

Molecular epidemiology of Acinetobacter baumannii bloodstream isolates obtained in the United States from 1995 to 2004 using rep-PCR and multilocus sequence typing.

Using a repetitive-sequence-based (rep)-PCR (DiversiLab), we have molecularly typed Acinetobacter nosocomial bloodstream isolates (Acinetobacter baumannii [n = 187], Acinetobacter pittii [n = 23], and Acinetobacter nosocomialis [n = 61]) obtained from patients hospitalized in U.S. hospitals over a 10-year period (1995-2004) during a nationwide surveillance study (Surveillance and Control of Pathogens of Epidemiological Importance [SCOPE]). Patterns of A. baumannii rep-PCR were compared to those of previously identified international clonal lineages (ICs) and were further investigated by multilocus sequence typing (MLST) to compare the two typing methods. Forty-seven of the A. baumannii isolates clustered with the previously defined IC 2. ICs 1, 3, 6, and 7 were also detected. The remaining 81 isolates were unrelated to the described ICs. In contrast, A. pittii and A. nosocomialis isolates were more heterogeneous, as determined by rep-PCR. Our MLST results were in good correlation with the rep-PCR clusters. Our study confirms previous data indicating the predominance of a few major clonal A. baumannii lineages in the United States, particularly IC 2. The presence in the United States of A. baumannii ICs 1, 2, and 3 from as early as 1995 suggests that global dissemination of these lineages was an early event.