Characterisation of anticancer peptides at the single-cell level.

The development of efficacious anticancer therapeutics is difficult due to the heterogeneity of the cellular response to chemotherapy. Anticancer peptides (ACPs) are promising drug candidates that have been shown to be active against a range of cancer cells. However, few ACP studies focus on tumour single-cell heterogeneities. In order to address this need, we developed a microfluidic device and an imaging procedure that enable the capture, monitoring, and analysis of several hundred single cells for the study of drug response. MCF-7 human breast adenocarcinoma cells were captured in hydrodynamic traps and isolated in individual microchambers of less than 100 pL volume. With pneumatic valves, different sets of microchambers were actuated to expose the cells to various drugs. Here, the effect of three membranolytic ACPs - melittin, aurein 1.2 and aurein 2.2 - was investigated by monitoring the efflux of calcein from single MCF-7 cells. The loss of membrane integrity was observed with two different strategies that allow either focusing on one cell for mechanistic studies or parallel analysis of hundreds of individual cells. In general, the device is applicable to the analysis of the effect of various drugs on a large number of different cell types. The platform will enable us in the future to determine the origin of heterogeneous responses on pharmacological substances like ACPs within cell populations by combining it with other on-chip analytical methods.

Release from apoptosis correlates with tumor progression in the AKR lymphoma.

Disturbance of apoptosis is an established factor in tumorigenesis. The role of apoptosis in tumor progression is not yet clear. In the present study we compared the tendency to spontaneous apoptosis (and the proliferative capacity) of tumor cells derived from primary (PT) and metastatic tumor (MT) cells of several AKR lymphoma variants. Apoptosis-related gene expression was also compared. Our results indicate that release from apoptosis has a role in the tumor progression of this T cell lymphoma. At the cellular level, a markedly lower apoptotic tendency was observed in MT than in PT cells. The existence of macrophages only in PT also supports the presence of apoptotic cells in local but not in MTs. By contrast, proliferative capacity does not determine tumor aggressiveness in this system. At the molecular level, we found a higher staining intensity for bcl-2 in MT than in PT cells, suggesting that bcl-2 might be responsible for the reduced apoptosis in MT compared to PT cells. Evidence for p53 overexpression was found in the MT cells of one of the variants but in none of the PT. Comparison of Fas receptor, unexpectedly showed an increased expression in MT versus PT cells, possibly indicating resistance to Fas-induced apoptosis in the MT cells.

Multidisciplinary forensic approach--investigation of a neonaticide and alleged concomitant rape.

In the case presented, a young woman claimed to have spontaneously aborted a 30-weeks old fetus conceived after being raped by her cousin. The police investigation was centred on a probable neonaticide, mitigated by a possible rape. In Israel, the crime of rape carries a heavy punishment similar to that of first degree murder. Thus the implementation of the most recent scientific forensic techniques is of paramount importance in charging or clearing a suspected rapist. The skeletonized fetal remains were found four months after the reputed abortion. The alleged neonaticide was supported by the results of the forensic clinical and anthropological examinations, while the rape accusation was dismissed by the results of the molecular biology identification of the skeletonized fetal remains. The aim of this report is to encourage the forensic community to apply a multi-disciplinary approach, thus maximizing the decision making competence of the courts of law.

Role of immune response as determinant of tumor progression in function of host age in the B16 melanoma.

Aging constitutes the major cause for the development of most neoplastic diseases. However, tumors in aged people present with a lower degree of aggressiveness than in young patients. The reasons for this paradoxical behavior are not clear. We attempted to verify whether the immune system has a role in the relation between host age, immune response and tumor progression. We compared the growth rate of B16 melanoma and a highly malignant variant, the B16/Col/R, in young and aged mice that have or have not undergone splenectomy. The following results were obtained: (1) Splenectomy stimulated growth in the parental melanoma in both young and aged mice, indicating a protective role of the spleen against this tumor at all ages; (2) Spleen ablation provoked inhibition of the highly-metastatic variant growth in young mice, suggesting a stimulatory role of the spleen in this case; (3) By contrast, in aged mice inoculated with the B16/Col/R variant, splenectomy enhanced tumor growth, indicating a defensive role of the spleen. Age favors a positive host response against the aggressive clone of the melanoma. Differential host response in young versus aged mice can explain, in this tumor system, the difference in tumor progression rate as a function of age.

Basement-membrane thickening of the vocal cords in sudden infant death syndrome.

Sudden infant death syndrome remains the leading cause of death in infants between the ages of 1 month and 1 year. Diagnosis at autopsy is usually reached by process of elimination, as no obvious cause of death is recognized. The larynges of 23 sudden infant death syndrome victims, as well as 6 infants and 3 fetuses who died of other known causes, were examined in this study. While thickening of the basement membrane of the vocal cords was apparent in all sudden infant death syndrome victims, no such finding was present in the larynges of infants and fetuses dying of other causes. Thus, it is suggested that basement-membrane thickening may serve as a diagnostic tool for identification of sudden infant death syndrome at autopsy.

Forensic radiology.

Imaging techniques are a powerful tool in forensic science. Medical examiners and forensic anthropologists are less versed in the finer points of radiology than radiologists; nevertheless they are required to interpret findings from imaging studies to further medico-legal investigations. The forensic investigator often should call upon the radiologist whose expertise might prove invaluable in forensic consultations. The radiologist should be aware of the importance of storing radiographs over prolonged periods of time and of efficient record keeping methods, because various legal problems may require the radiographs for additional interpretation or for their presentation in court. Some of the main issues that might be encountered in forensic radiology are discussed in this review.

Gingival hyperplasia caused by nifedipine. Histopathologic findings.

Thirty-four cardiac patients, treated with nifedipine (Adalat, Procardia) during a period of 1 year or more, developed slight to severe gingival epithelial hyperplasia, and five cases presented nodular overgrowth of the gingiva. In terms of histopathologic and clinical morphology, nifedipine-induced gingival hyperplasia is similar to that known to be caused by phenytoin (Dilantin). Both drugs have the ability to alter calcium metabolism. We believe that the hyperplastic condition is related to calcium imbalance.

The microscopic structure and function of the vascular retrodiscal pad of the human temporomandibular joint.

The highly vascular retrodiscal pad attaches the articular disc of the temporomandibular joint to osseous structures posterior to it. There is debate as to whether or not the pad includes erectile tissue. Histological examination of 11 retrodiscal pads revealed thick-walled muscular arteries, thin-walled veins and numerous vascular sinuses, which is different from the erectile tissue of the corpora cavernosa. Its histological features and position within the joint suggest that the retrodiscal pad may function differently from other human joints due to: (1) the large amount of distraction it undergoes relative to its size and (2) its location anterior to the ear.

Biological behavior and cell properties of new AKR lymphoma malignancy variants.

The AKR lymphoma-leukemia is a T lymphocyte neoplasm, most suitable as a model for human T cell malignancies. We have been interested in the process of tumor progression in the AKR lymphoma system. In the present study, two newly isolated variants, the TAU-42 and TAU-44, were characterized with respect to their biological behavior, by comparing them to a previously studied low-malignancy variant, the TAU-39. While the TAU-44 variant formed large s.c. local tumors, the TAU-42 variant formed only small growths or none at all. The TAU-42 lymphoma was found to have the highest malignant potential: it displayed very marked dissemination to spleen, lymph nodes, liver and lungs. The TAU-44 variant had an intermediate degree of metastatic potential but presented a predilection for spread to lymph nodes and spleen and was sometimes found to metastasize to peculiar organs, such as heart and pancreas. Cells derived from the different lymphoma variants varied in their immunophenotype: the highly malignant variant cells expressed more CD4 antigen than the low-malignancy one. The opposite was observed with regard to CD8. The variant cells also differed in their migrating capacity, the more malignant one exhibiting a higher motile activity. Studies on the tumor progression model of AKR lymphoma might contribute to the elucidation of the features determining the aggressiveness of T lymphocytic malignancies.

Intramedullary spinal cord sarcoidosis.

A case of histologically proven intramedullary spinal cord sarcoidosis diagnosed antemortem is presented. The patient manifested symptoms referable only to the spinal cord simulating a tumor--progressive tetraparesis. The histologic diagnosis of sarcoidosis was established through exploratory laminectomy. Surgical intervention is the only way to rule out a malignant process in case of sarcoidosis limited to the spinal cord.

Sensitivity to macrophages decreases with tumor progression in the AKR lymphoma.

Resistance to immune reactions, innate or acquired, may be one of the mechanisms responsible for the progression of tumors. We have, indeed shown higher numbers of macrophages surrounding low- as compared to high-malignancy cells. In the present study we examined the level of cell surface molecules known to determine sensitivity to macrophages, namely galactose (GAL) and sialic acid (SA) residues. A histochemical assay for identification of SA by electron microscopy showed a higher cell surface content on metastatic (MT) than on primary (PT) tumor cells. The FACS data seen with fluorescent lectins showed a higher binding of Sambucus nigra agglutinin, which identifies SA attached to terminal GAL in -2.6 or -2.3 linkage, in MT than in PT cells. Binding of Maakia amurensis lectin (MAL-1), which identifies SA at position 3 of GAL, showed that the MT cells contain two subpopulations, one binding more MAL-1 and another less. Cell sorting showed a more aggressive behavior of the first population. The comparison of Peanut agglutinin (PNA) binding, which identifies GAL, demonstrated a decreased amount of PNA receptors in MT as compared to PT cells. Western blot analysis of the membranal proteins with different lectins, identified 3 sialylated glycoproteins. The 88 kDa glycoprotein had no significance for metastatic potential. The 130 kDa glycoprotein was higher in MT than on PT cells. The 220 kDa glycoprotein was practically present only on MT cells. The tendency observed was of a higher level of membranal glycoconjugates terminally sialylated with subterminal galactose residues, inMT cells as compared to PT cells. This may explain the recently found decrease in apoptotic cell death with increasing aggressiveness of the AKR lymphoma and suggests a lower sensitivity to macrophages with tumor progression. Treatment based on the reduction in sialic acid content might render the tumor cells more vulnerable to macrophages. We found, indeed, that Wheat germ agglutinin (WGA) injected in vivo, exerted an inhibitory effect on growth of the lymphoma. We found moreover that WGA-treated tumor cells were more sensitive than nontreated cells to macrophages in vitro.

Imprint cytology in the intraoperative diagnosis of malignant melanoma.

Imprints were prepared from 73 pigmented skin lesions, 19 of which were diagnosed as malignant melanoma. The cytologic findings in malignant melanoma, large clusters of cells with loss of cellular cohesiveness and large pleomorphic nuclei, were positive in 17 of the 19 cases. In other malignant and benign pigmented lesions the few cells adherent to the glass showed characteristic cytologic features of the particular lesion. Imprint cytology seems to be a valuable adjunct to the examination of frozen sections for the intraoperative diagnosis of malignant melanoma.

Positive identification by means of trabecular bone pattern comparison.

Positive identification of human remains is often achieved by comparing antemortem and postmortem radiographs. Usually, radiographs contain a number of markers that can serve as reference for comparison, one of these markers is the trabecular bone pattern depicted in the roentgenograms. In the present case, densitometric analysis of the trabecular bone pattern was used as the sole means of identification. Later on, two other methods confirmed the original positive identification.

Suicidal terrorist bombings in Israel--identification of human remains.

Positive identification of human remains is one of the most important tasks in mass disaster investigations. Religious and jurisdictional demands in Israel, require the identification process to be completed in the shortest possible time. In the 18 suicidal terrorist bombings that took place in Israel between the years 1993-1996, 127 victims and 19 perpetrators were killed, and their severely fragmented bodies were identified within 24 h. The efficient completion of the identification endeavor was enabled by the implementation of a variety of techniques and the close collaboration in the investigation between the different emergency and forensic agencies. This paper presents the mass disaster identification policy and techniques currently used in Israel. The importance of an interdisciplinary approach for the identification of extremely fragmented human remains from mass disasters and the creation of a central data bank of fingerprints and genetic markers is emphasized.

Quantitative assessment of trabecular bone pattern identification.

The results of the research described in this paper demonstrate that the trabecular architecture is unique to each individual and stable enough to be used as a forensic marker for positive identification of human remains. The trabecular bone architecture depicted on radiographs is often used as an individualizing forensic marker for positive identification of human remains. The aim of the present study was to ascertain the reliability of the trabecular pattern in forensic identification. The trabecular pattern is potentially the best radiographic forensic marker since its presence on a radiograph doesn't depend on a previous pathology or traumatic event. A sample of 305 radiographs of the left wrist of 103 postmenopausal women was studied using an image analyzer. The uniqueness and stability over time of the trabecular architecture was examined by creating line maps or "densitographs" of the ultra-distal point of the radius of each roentgenogram. Pearson's correlation coefficients were calculated for all possible combinations of pairs of radiographs. The correlation coefficient of pairs of radiographs of the same individual, taken at different times (2 to 6 years apart), was always higher than 0.72, while the correlation coefficients of radiographs of different individuals was always below 0.62.

Fingerprinting the deceased: traditional and new techniques.

Identification of the deceased is an important task in medicolegal investigations. Fingerprints rank as the most widely used identification method, although obtaining the prints from the cadaver is not always easily accomplished. Various techniques for fingerprinting decomposed, mummified and burn victims have been suggested in the literature. In the present review, the diverse fingerprinting procedures implemented for cadavers in various conditions, with an emphasis on mummified fingers, are presented.

Estimation of age in adolescents --the basilar synchondrosis.

The state of fusion of the basilar synchondrosis as a biological age indicator was assessed in a sample of 91 cadavers of both sexes whose ages ranged between 8 and 26 years. The correlation between the degree of closure and chronological age was investigated. Although the female population sample was very small (n = 21), the data indicate a tendency of differences in age between the "open" and "closed" groups. In the male population (n = 70), no significant differences were detected between the "open" and "closed" categories; in fact, the mean age of the two groups was the same (p = 0.9). These findings indicate that the stage of fusion of the basilar synchondrosis is not a good indicator of age in male cadavers, while in females the feature could be useful when estimating age of unknown human remains, although further investigation on a larger sample is advocated.

Improved method for shooting distance estimation. Part III. Bullet holes in cadavers.

An improved method to estimate firing distance on human body surfaces is described. The novel part of the method includes a chemical test in addition to the traditional visual and microscopic examinations of the gunshot wounds. This chemical test consists of a transfer of the gunpowder residues from the area of a gunshot wound to an adhesive lifter; the residues are then visualized as total nitrite after alkaline hydrolysis by the Modified Griess Test (MGT). When cadavers are in an advanced stage of decomposition or when gunshot wounds are in hairy areas, the information obtained by this chemical test can be crucial for shooting distance evaluation. In other cases it may improve the accuracy of the examination. In some cases, the results obtained by this test may assist in the discrimination between entrance and exit gunshot wounds.

Marine taphonomy: adipocere formation in a series of bodies recovered from a single shipwreck.

Taphonomy of marine environments has been studied mostly from individual cases. The formation of adipocere, or "grave-wax," is an important indicator of the postmortem interval. In the present paper, the conditions and the timing of adipoceric formation are observed in a series of 15 cadavers recovered at different times, over a period of 433 days, from the same contained environment. Initial foci of adipocere on the subcutaneous tissue of the cadavers were detected as early as 38 days from the time of immersion in cold (10-12 degrees C) sea water. The discrepancies between our findings and previous reports on the correlation between time since death and decomposition stages in marine environments are discussed.

Trauma and identification of victims of suicidal terrorism in Israel.

The postmortem examination and identification procedures performed by medical and law enforcement personnel involved in mass disaster management in Israel are reported. The Israel National Police, the Israel Defense Forces, and the L. Greenberg Institute of Forensic Medicine's experts examined 171 victims who died in 21 incidents of suicidal terrorism. The trauma sustained by the victims and perpetrators of suicidal bombings included complete body disruption and explosive, flying missile, and blast injuries. The modus operandi of the perpetrators, reconstructed from the distribution and type of injury of the victims, is discussed. Fifty-five victims perished in open space bombings and 91 inside buses. All perpetrators of these bombings died at the time of the incident regardless of their location. Identification of the victims was achieved using fingerprints, dental records, medical intervention signs, anatomic variation, genetic profile, and personal recognition. Prompt identification of the perpetrators allowed speedy apprehension of the accomplices and prevention of similar attacks. Collaboration between the different forensic, military, and law enforcement teams increased the efficiency of disaster management efforts.