Study on the performance of different craniofacial superimposition approaches (II): Best practices proposal.

Craniofacial superimposition, although existing for one century, is still a controversial technique within the scientific community. Objective and unbiased validation studies over a significant number of cases are required to establish a more solid picture on the reliability. However, there is lack of protocols and standards in the application of the technique leading to contradictory information concerning reliability. Instead of following a uniform methodology, every expert tends to apply his own approach to the problem, based on the available technology and deep knowledge on human craniofacial anatomy, soft tissues, and their relationships. The aim of this study was to assess the reliability of different craniofacial superimposition methodologies and the corresponding technical approaches to this type of identification. With all the data generated, some of the most representative experts in craniofacial identification joined in a discussion intended to identify and agree on the most important issues that have to be considered to properly employ the craniofacial superimposition technique. As a consequence, the consortium has produced the current manuscript, which can be considered the first standard in the field; including good and bad practices, sources of error and uncertainties, technological requirements and desirable features, and finally a common scale for the craniofacial matching evaluation. Such a document is intended to be part of a more complete framework for craniofacial superimposition, to be developed during the FP7-founded project MEPROCS, which will favour and standardize its proper application.

Study on the performance of different craniofacial superimposition approaches (I).

As part of the scientific tasks coordinated throughout The 'New Methodologies and Protocols of Forensic Identification by Craniofacial Superimposition (MEPROCS)' project, the current study aims to analyse the performance of a diverse set of CFS methodologies and the corresponding technical approaches when dealing with a common dataset of real-world cases. Thus, a multiple-lab study on craniofacial superimposition has been carried out for the first time. In particular, 26 participants from 17 different institutions in 13 countries were asked to deal with 14 identification scenarios, some of them involving the comparison of multiple candidates and unknown skulls. In total, 60 craniofacial superimposition problems divided in two set of females and males. Each participant follow her/his own methodology and employed her/his particular technological means. For each single case they were asked to report the final identification decision (either positive or negative) along with the rationale supporting the decision and at least one image illustrating the overlay/superimposition outcome. This study is expected to provide important insights to better understand the most convenient characteristics of every method included in this study.

Soft tissue thickness values for black and coloured South African children aged 6-13 years.

In children, craniofacial changes due to facial growth complicate facial approximations and require specific knowledge of soft tissue thicknesses (STT). The lack of South African juvenile STT standards of particular age groups, sex and ancestry is problematic. According to forensic artists in the South African Police Service the use of African-American values to reconstruct faces of Black South African children yields poor results. In order to perform a facial approximation that presents a true reflection of the child in question, information regarding differences in facial soft tissue at different ages, sexes and ancestry groups is needed. The aims of this study were to provide data on STT of South African Black and Coloured children and to assess differences in STT with respect to age, sex and ancestry. STT was measured using cephalograms of South African children (n=388), aged 6-13 years. After digitizing the images, STT measurements were taken at ten mid-facial landmarks from each image using the iTEM measuring program. STT comparisons between groups per age, sex and ancestry were statistically analyzed. The results showed that STT differences at lower face landmarks are more pronounced in age groups per ancestry as opposed to differences per age and sex. Generally, an increase in STT was seen between 6-10 year old groups and 11-13 year old groups, regardless of ancestry and sex, at the midphiltrum, labiale inferius, pogonion, and beneath chin landmarks. This research created a reference dataset for STT of South African children of Black and Coloured ancestry per age and sex that will be useful for facial reconstruction/approximation of juvenile remains.

Characterization of newly established testicular peritubular and prostatic stromal cell lines: potential use in the study of mesenchymal-epithelial interactions.

Testicular peritubular and prostatic stromal cells produce extracellular matrix elements and paracrine factors that modulate the cytodifferentiation and function of the corresponding epithelial cells. The present paper describes the establishment and characterization of five rat testicular cell lines with peritubular characteristics and one prostatic stromal cell line. Four peritubular cell lines were isolated after transfection of a mixed peritubular-Sertoli cell culture with a v-myc-containing plasmid. The same immortalization procedure applied to prostatic stromal cells yielded one cell line. An additional testicular cell line arose by spontaneous immortalization during serial subculture. Except for one testicular cell line (RTC-8T1), the morphology of all of the immortalized cell lines strongly resembled that of primary cultures of peritubular and stromal cells. Flow cytometric analysis demonstrated that all cell lines scored positive for alpha-smooth muscle isoactin and negative for cytokeratins, confirming their myofibroblast-like nature. None of the cell lines, however, stained positive for alkaline phosphatase, and androgen receptor expression was also lost. Typical Leydig cell characteristics, such as steroidogenesis, and Sertoli cell markers, such as transferrin secretion, were absent. Coculture of the cell lines with Sertoli cells resulted in the formation of tubular structures. A cell attachment assay and an enzyme-linked immunosorbent assay for fibronectin confirmed the production of extracellular matrix elements by all of the established cell lines. Media conditioned by the cell lines stimulated Sertoli cell transferrin production. The active principle was partially purified and resembled the P-MOD-S-like factors produced by primary cultures of peritubular and stromal cells. It is concluded that the immortalized cell lines have retained several of the characteristics of primary cultures of peritubular and stromal cells and may be useful for further studies on mesenchymal-epithelial interactions in testis and prostate.

Generation and characterization of mouse microglial cell lines.

A murine cell line (MMGT1) has been established after transfection of primary microglial cell cultures with a v-myc-containing plasmid. This cell line was comparable with primary microglial cells with respect to morphology, presence of acetylated low density lipoprotein receptor, non-specific esterase, CD63, major histocompatibility complex antigens and CD11, and binding for Ricinus communis agglutinin. Primary microglia as well as MMGT1 cells were negative for glial fibrillary acidic protein. Different MMGT1 strains were obtained after subcloning, two of which resembled histiocytes (F4/80 and BM-8). These cell strains, MMGT12 and 16, were able to opsonize latex beads, and could be induced by endotoxins (LPS) to secrete TNF-alpha, IL-1, IL-6, TGF-beta, and EGF. The other subclones had intermediate (MCA519, ER-MP20) or mixed macrophage characteristics and did not react to endotoxin by an increase in TNF-alpha, IL-1, and TGF-beta. Our newly established murine microglial lines may prove to be useful models to study inflammation and repair in the brain.

Secretion of steroids, growth factors, and cytokines by immortalized mouse granulosa cell lines.

The expression and function of gonadotropin receptors, and the secretion of steroids, transferrin, and cytokines were investigated in three immortalized (single transfection with v-myc) mouse granulosa cell lines (GRM01, GRM01L, and GRM02). A dose-dependent increase in progesterone production was obtained in GRM01 and GRM02 cells after addition of LH, FSH, modulators of the adenylate cyclase enzyme system, and cAMP analogues. The LH-induced release of progesterone was already detectable in GRM02 cells after 8 h and was related to incubation time and cell number. Both epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) induced the secretion of progesterone in GRM02 cells, while no effect was obtained with TGF beta. LH receptor concentration was highest in the GRM02 cell line. FSH receptor mRNA was visualized in GRM01 and GRM02 cells. Aromatase activity in GRM02 cells was induced by androgens and inhibited by aromatase inhibitors. Whereas all cell lines were able to secrete transferrin, only in GRM01 cells was transferrin secretion increased significantly by LH. FSH did not affect transferrin secretion in the three cell lines, in contrast to forskolin or 8-bromo-cAMP. The immortalized mouse granulosa cell lines were able to express and release several growth factors. The expression and secretion of activin, inhibin, TGF beta, EGF, TGF alpha, insulin-like growth factor II, fibroblast growth factor (acidic and basic), platelet-derived growth factor, and interleukin-6 suggest an autocrine or paracrine role for these factors in follicular differentiation and function. In conclusion, these cells, derived from mural granulosa cells and immortalized in a preovulatory state, can be used to study granulosa cell physiology or to study the role of granulosa cells and their derivatives in the process of follicular maturation, fertilization, and early embryonic development.

Characterization of immortalized mouse granulosa cell lines.

Cell cultures of primary mouse granulosa cells were transfected with a v-myc-containing plasmid, and the resulting stable cell lines were tested for their steroidogenic properties and physiologic status. Granulosa cells were obtained from 22-day-old NMRI mice injected with 8 IU pregnant mare serum gonadotropin i.p. 2 days earlier. In Passage 1 the cells were transfected with pSVv-myc using calcium phosphate precipitation or lipofectin. The 3 beta- and 17 beta-hydroxy steroid dehydrogenase activity was visualized in control cultures. The three cell lines obtained have been in culture for over 1 yr and have been subcultured for more than 90 passages. The cell line GRM01, with a doubling time of 37 +/- 3 h and a diploid modal chromosome number, produced progesterone, estradiol, as well as inhibinlike and activinlike material under basal conditions. A combination of follicle-stimulating hormone and luteinizing hormone was able to increase the secretion of progesterone. GRM01L, a fast growing clone of the GRM01 line with a doubling time of 10 +/- 1 h, retained only the capacity to produce activinlike material and transforming growth factor-beta, and it was the only one with a tumorigenic capacity. Epidermal growth factor, insulin, and interleukin-6 were able to induce the [3H]thymidine incorporation into DNA in these two cell lines. GRM02, with a doubling time of 36 +/- 2 h and a hypertriploid modal chromosome number, produced progesterone and activinlike and inhibinlike material. Follicle-stimulating hormone and luteinizing hormone were able to enhance the secretion of progesterone. For this cell line, only insulin was shown to induce [3H]thymidine incorporation into DNA.

Establishment and characterization of a human ovarian neoplastic cell line, DO-s.

A permanent human neoplastic cell line, DO-s, was established from ascites of a patient with a well-differentiated mucinous cyst-adenocarcinoma of the ovary. This cell line grew as vermiform, floating colonies of epithelial cells in culture. The karyotype of DO-s was of a human female; the chromosome number ranged from 54 to 66 with several abnormalities, mainly trisomy. Epithelial-like character was confirmed by transmission electron microscopy and by the presence of cytokeratin. Inoculation of DO-s cells i.p. or s.c. in athymic nude mice resulted in, respectively, ascites and xenografts. Light and electron microscopical analysis of cultured cells and xenografts demonstrated that the cell line was derived of a mucinous adenocarcinoma biopsy. Tumor-associated antigens, cancer antigen 125 (CA 125), human milk fat globulin, and human placental alkaline phosphatase were expressed by cells in culture and in xenografts. Modulation of the antigens, CA 125 and human milk fat globulin, occurred in DO-s cells growing in athymic mice. Biochemical, immunohistochemical, and histochemical analysis showed that more than 50% of the alkaline phosphatase isoenzymes present in DO-s cells had the characteristics of human placental alkaline phosphatase and placental alkaline phosphatase-like alkaline phosphatase (AP), but fractions of intestinal AP and nonspecific AP (bone-liver-kidney) were also present. The expression of AP isoenzymes could be induced by an enhancement of the serum supplement in the culture media, and by dexamethasone, sodium butyrate, and bromodeoxyuridine. This line will be a valuable tool in studying the therapeutic effects of antibodies to tumor-associated antigens or other agents for ovarian cancer.

Immunohistochemical localization of placental alkaline phosphatase, carcinoembryonic antigen, and cancer antigen 125 in normal and neoplastic human lung.

Human placental alkaline phosphatase (HPLAP), carcinoembryonic antigen (CEA), and cancer antigen 125 (CA 125) were localized immunohistochemically in paraffin sections of normal lung tissue from 16 patients, using monoclonal antibodies and an indirect avidin-biotin-peroxidase staining procedure. HPLAP and CEA were present in epithelial cells of respiratory bronchioli and alveolar type I pneumocytes. CEA was also observed in the tracheal, bronchial, and bronchiolar epithelium. CA 125 was present in the tracheal, bronchial, bronchiolar, and terminal bronchiolar epithelium; in the tracheal and bronchial glands; and in the pleural mesothelium. Normal and hyperplastic type II pneumocytes were negative for HPLAP, CEA, and CA 125 but were histochemically positive for nonspecific alkaline phosphatase. Fetal lung tissue between 11 and 15 weeks of gestation was negative for HPLAP, CEA, and CA 125. The fetal tracheal and bronchial epithelium, tracheal glands, and pleural mesothelium were positive for CA 125. For ten malignant pulmonary tumors investigated, HPLAP staining was observed in five, CEA in nine, and CA 125 in seven. The localization of HPLAP, CEA, and CA 125 in apparently normal constituents of all pulmonary specimens is in disagreement with the concept that the expression of these substances in the lung is indicative of abnormal cellular activity.

Placental alkaline phosphatase and cancer antigen 125 in sera of patients with benign and malignant diseases.

Human placental alkaline phosphatase (hPLAP; EC 3.1.3.1), cancer antigen 125 (CA 125), and carcinoembryonic antigen (CEA) were determined in sera of patients with malignant and nonmalignant disorders. For CA 125 we used two different commercial assay systems, based on the same monoclonal antibody. hPLAP had the same sensitivity (20%) as CA 125 for detecting non-ovarian neoplasia, whereas that of CEA was 45%. For detecting ovarian cancer CA 125 (Cis kit) was slightly more sensitive (50%) than hPLAP (45%), much more than CEA (10%). hPLAP was increased in sera of 2% of patients with nonmalignant disorders, CA 125 in 23%, and CEA in 18%. hPLAP was increased in only one of 10 diabetic patients and two of 50 patients on chronic renal dialysis. CA 125 and CEA were respectively increased in 45% and 23% of all liver pathologies studied and in 12% and 17% of patients with renal insufficiency. The sensitivity of hPLAP for detecting ovarian cancer is slightly inferior to that of CA 125, but its specificity is much higher. We found the Abbott system for CA 125 to be more sensitive than the Cis system.

Photoaffinity labeling of the hemolymph juvenile hormone binding protein of Manduca sexta.

A synthetic analogue of the insect juvenile hormone (JH) III, 10,11-epoxy[10-3H]farnesyl diazoacetate [( 3H]-EFDA), binds to several proteins in a partially purified preparation of hemolymph protein from fourth instar larvae of Manduca sexta when irradiated with UV light. Approximately 80% of this binding could be inhibited by the addition of excess unlabeled JH I. To compare the relative affinity of EFDA for the juvenile hormone binding protein (JHBP) with that of the various JH homologues, the ability of unlabeled EFDA and JH homologues to displace [3H]JH I from binding sites was measured. The relative affinities were EFDA greater than JH I greater than JH II greater than JH III. When Scatchard analysis of the binding of [3H]EFDA or [3H]JH I to the larval JHBP was performed, an estimated apparent KD of 4.5 X 10(-8)M was found for EFDA, whereas for JH I a slightly higher KD of 8.8 X 10(-8) M was calculated. To determine if [3H]EFDA bound at the JH I binding site, displacement of [3H]JH I from the JHBP complex with unlabeled JH I, JH II, and JH III was compared to the displacement of [3H]EFDA with the same homologues. The results demonstrated that the photoaffinity label bound covalently at the JH I binding site on the hemolymph binding protein of Manduca sexta. Fluorescence autoradiography of [3H]EFDA photoaffinity labeled proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that [3H]EFDA bound covalently to two major proteins in the absence of JH I.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of injected [3H]ecdysone in male Sarcophaga bullata (Diptera).

Within 30 min after injection, [3H]ecdysone was rapidly partially metabolized to ecdysterone and other ecdysteroids. After 8 hr most (99%) of the tritiated material had disappeared from the hemolymph. In testes, the predominant ecdysteroid appeared to be ecdysterone but no accumulation occurred. Comparison of the dynamics of the ecdysone metabolism in abdomens and head-thorax sections showed that in the head-thorax ecdysterone was the major component, whereas in abdomens the major metabolites were highly polar products (HPP). However, the total amount of label was nearly the same in both parts. Two groups of HPP have been isolated from the abdomen fractions without testes: HPP B and HPP C. Only HPP B could be hydrolyzed by enzymes and seemed at least to contain glucuronides, beta-glucosides, and sulfate conjugates. After 4 hr most of the tritiated ecdysteroids were found in the fecal material. No male-specific metabolites have been discovered.

Moulting hormone, juvenile hormone and the ultrastructure of the fat body of adult Sarcophaga bullata (Diptera).

In the ovoviviparous fly, Sarcophaga bullata, vitellogenesis in cyclic; a process reflected in ultrastructural changes in the fat body cells and oenocytes. At eclosion the larval fat body has not yet completely disappeared. During vitellogenesis the fat body cells are specialized for intensive protein synthesis showing a very extensive RER and numerous invaginations of the plasma membrane. These features disappear when the eggs descend into the oviducts to complete embryogenesis. The predominant feature of the oenocytes is their very prominent SER. The fat body cells of the males are never as specialized for protein synthesis as those of the females. Feeding of ecdysterone to males for 3 or more days induces a rather extensive subcellular apparatus for protein synthesis, i.e., invaginations of the plasma membrane and an extensive RER. Juvenile hormone is completely ineffective in this respect. Both ecdysterone and juvenile hormone have pronounced but different effects on the oenocytes of males.