Determination of drugs of abuse in bovine dentin using liquid chromatography-electrospray ionization tandem mass spectrometry.

Drugs deposited in human teeth are well preserved; the spectrum of toxicological investigations may therefore be supplemented by an analysis method for drugs in teeth. A liquid chromatography-electrospray ionization tandem mass spectrometry assay for the detection and quantification of basic drugs of abuse in bovine dentin samples was developed and validated. The drugs and metabolites amphetamine, methamphetamine, methylenedioxymethylamphetamine, methylenedioxyethylamphetamine, codeine, morphine, cocaine and benzoylecgonine were extracted from 50 mg ground dentin powder by ultrasonication for 60 min in methanol 3 times. The extracts were analyzed on a triple-quadrupole mass-spectrometer in multiple reaction monitoring mode. The method was validated and proved to be accurate, precise, selective, specific and stable with good linearity within the calibration range and a lower limit of quantification of 10 to 20 pg/mg. To artificially load bovine dentin samples with drugs, the natural process of de- and remineralization in the oral cavity was mimicked by a pH-cycling experiment. The artificially drug-loaded dentin samples showed drug concentrations of 20 to 80 pg/mg. The method can be applied in further in vitro experiments as well as in post-mortem cases, especially where limited sample tissue is available.

The German national registry for primary immunodeficiencies (PID).

In 2009, a federally funded clinical and research consortium (PID-NET, http://www.pid-net.org) established the first national registry for primary immunodeficiencies (PID) in Germany. The registry contains clinical and genetic information on PID patients and is set up within the framework of the existing European Database for Primary Immunodeficiencies, run by the European Society for Primary Immunodeficiencies. Following the example of other national registries, a central data entry clerk has been employed to support data entry at the participating centres. Regulations for ethics approvals have presented a major challenge for participation of individual centres and have led to a delay in data entry in some cases. Data on 630 patients, entered into the European registry between 2004 and 2009, were incorporated into the national registry. From April 2009 to March 2012, the number of contributing centres increased from seven to 21 and 738 additional patients were reported, leading to a total number of 1368 patients, of whom 1232 were alive. The age distribution of living patients differs significantly by gender, with twice as many males than females among children, but 15% more women than men in the age group 30 years and older. The diagnostic delay between onset of symptoms and diagnosis has decreased for some PID over the past 20 years, but remains particularly high at a median of 4 years in common variable immunodeficiency (CVID), the most prevalent PID.

Observation of graphene bubbles and effective mass transport under graphene films.

Mechanically exfoliated graphene mounted on a SiO2/Si substrate was subjected to HF/H(2)O etching or irradiation by energetic protons. In both cases gas was released from the SiO2 and accumulated at the graphene/SiO2 interface resulting in the formation of "bubbles" in the graphene sheet. Formation of these "bubbles" demonstrates the robust nature of single layer graphene membranes, which are capable of containing mesoscopic volumes of gas. In addition, effective mass transport at the graphene/SiO2 interface has been observed.

Embryology of dolphins. Staging and ageing of embryos and fetuses of some cetaceans.

Comparative embryology has been neglected in the past decades. In our opinion the decreased interest has been caused by the fact that the time factor has not sufficiently been respected. Therefore we used the Staging and Ageing Method (Sterba 1999) to determine the ontogenetic age of embryos and to state the time tables and time relations of various ontogenetic events. We examined 167 embryos and fetuses of four dolphin species (collection Dr. Senckenbergische Anatomie, Frankfurt am Main). The study of intrauterine development and growth while consistently respecting the time factor enabled us to determine the time course of organogenesis of all organ systems. The resulting comparison with other mammals detected the time lag in ossification, retardation of odontogeny, the time of the origin and development of the fluke, dorsal fin and flipper, etc. We believe the Staging and Ageing Method serves for a much more efficient evaluation of existing embryological collections which very often contain embryos of unknown age.

Development of the cetacean nasal skull.

The adaptation of cetaceans to aquatic life habits is reflected, in their nasal region, in three marked changes from the original relations found in land mammals. These changes include (1) the loss of the sense of smell, (2) translocation of the nostrils from the tip of the rostrum to the vertex of the head, and (3) elongation of the anterior head to form a rostrum protruding far towards anterior. The morphogenetic processes taking place during embryogenesis of the nasal skull play a decisive part in the development of all these changes. The lateral parts of the embryonic nasal capsule, encompassing the nasal passages, change their position from horizontal to vertical. At the same time, the structures of the original nasal floor (the solum nasi) are shifted in front of the nasal passages towards the rostrum. The structures of the original nasal roof (the tectum nasi) and of the nasal side wall (the paries nasi) are translocated behind the nasal passages towards the neurocranium. The medial nasal septum (the septum nasi) mostly loses its connection to the nasal passages and is produced into a point protruding far towards anterior. The transformed embryonic nasal skull of the Cetacea can be divided into three sections: 1. The median structures. These include the cartilaginous structures, viz., the rostrum nasi, the septum interorbitale and the spina mesethmoidalis, which are accompanied by the dermal bones, the vomer and the praemaxillare. In adult cetaceans the rostrum nasi is mostly preserved as a robust cartilage of the skull, which may possibly serve as a sound transmitting structure of the sonar system, or it may be responsible for the sensing of water streams and vibrations. 2. The posterior side wall structures. These include the following cartilaginous structures that are mostly heavily reduced or mutually fused: the cupula nasi anterior, the tectum nasi, the lamina cribrosa, the paries nasi, the commissura orbitonasalis, the cupula nasi posterior, the processus paraseptalis posterior, the crista semicircularis, the frontoturbinale, the ethmoturbinale I and the maxilloturbinale. The cartilaginous structures are largely accompanied by the dermal bone, the maxillare. Of these embryonic elements, very little is preserved in adult cetaceans. The cartilages of the cupula nasi anterior form the variable skeleton around the nostrils. In Physeter the tectum nasi forms a very long cartilaginous bar that passes through the whole giant anterior head of the sperm whale as a structure accompanying the left nasal passage. 3. The anterior side wall structures. These include the cartilaginous structures, viz., the cartilago ductus nasopalatini, the cartilago paraseptalis, the processus lateralis ventralis and the lamina transversalis anterior, accompanied by the dermal bones, the praemaxillare and the vomer. These structures participate in the formation of the robust rostrum of the cetacean skull, and they are partly preserved even in adults in the form of the isolated ossa pararostralia (the Meckelian ossicles). The comparison of morphogeny of the nasal skull has also made it possible to draw certain conclusions on the phylogeny and systematics of Cetacea. Already the earliest embryonic stages permit us to discern weighty transformations of the original nasal skull of land mammals. These transformations are common to all embryos examined. This fact indicates a common origin of all Cetacea, which thus form a single monophyletic order. However, later embryonic stages show some different modifications of the nasal capsule according to which at least three major groups can be distinguished within the order Cetacea, probably ranking as superfamilies: Balaenopteroidea, Physeteroidea and Delphinoidea. Our observations, being in full accordance with other morphological, and embryological, as well as molecular biological results, suggest that the division of the order Cetacea into two suborders, Mysticeti and Odontoceti, is no longer tenable.

Causes of death in geriatric patients: a cross-cultural study.

BACKGROUND: The elderly are living longer and causes of death are shifting. At the same time, autopsy rate is at, or near, its lowest in history, compounded by an even lower interest in geriatric autopsies. Thus, the prevalent cause of death in this age group remains poorly studied. METHODS: In a retrospective study, the autopsy protocols of 440 70-year-old or older patients from the Houston Veterans Affairs Hospital and 321 80-year-old or older patients from the II*Institute of Pathology in Prague (Czech Republic) were reviewed in order to establish a correct cause of death. The autopsy diagnosis was correlated with the prosectors' description of pathological findings in the protocol. In questionable cases or discrepancies, the patient's clinical chart and/or the histological autopsy slides were also reviewed. RESULTS: The distribution of death by infections and cardiac disorders each accounted for one-third of all deaths. Congestive heart failure prevailed in the over 80-year-olds, and myocardial infarcts prevailed in the younger patients. The number of deaths due to malignancy dropped from 25% in those 70-79 years old to about 10% in the elder patients. Central nervous system disorders were frequent as an underlying disease, but were not common as a cause of death. The findings were similar in both series, thus supporting their accuracy. CONCLUSION: Our findings bring into question the accuracy of reported causes of death in the elderly. With increasing age, differences appear in the levels of mortality and morbidity for various disease categories. This study underlines the need for more baseline data for older people which can be obtained only by more and well-performed autopsies.

On the development of Cetacean extremities: II. Morphogenesis and histogenesis of the flippers in the spotted dolphin (Stenella attenuata).

Externally, the flippers of Cetacea resemble fish fins, but their internal structure is entirely mammalian. They show, however, some adaptative deviations from the typical pattern of the mammalian extremities, the most striking of which is an increased number of phalanges. The aim of this study is to describe the course of the development of flippers in the spotted dolphin (Stenella attenuata) and compare its features with other similar species from an evolutionary perspective. Early stages of flipper development were studied histologically. Differentiation of cartilaginous anlagens of the skeleton progresses proximodistally, condensation in digital rays being evident sooner than chondrogenesis in the carpal region. In one specimen, the temporary presence of cartilaginous rudiments of two carpal elements, which are not found in adults, was observed. At all examined stages, phalangeal number progressively increases up to (radial to ulnar) 3, 7, 7, 5, 3 in the most advanced stage. The reason for this condition is the specialised function of these limb-like structures. It is a classical example of convergence, in which mammalian extremities change their form to emulate the fin function. A similar condition is found in another group of originally terrestrial animals secondarily fully adapted to the aquatic mode of life-Ichyosauria (Reptilia).

On the development of Cetacean extremities: I. Hind limb rudimentation in the Spotted dolphin (Stenella attenuata).

The Cetacea are group of animals which have completely lost their hind limbs during the course of evolution as a result of their entirely aquatic mode of life. It is known, however, that during their embryonal period, the hind limb buds are temporarily present. The control mechanisms of this regression are not yet understood, and vestigial limbs can sometimes be found in adults. The aim of the present study is to describe the course of hind limb rudimentation during prenatal development of Stenella attenuata (Spotted dolphin) at tissue and cell levels and compare the results with other natural or experimentally induced amelias. Hind limb buds of dolphin embryos, CRL 10-30 mm, were examined histologically. Before total disappearance, they show histodifferentiation comparable with other mammals. Initially, they form the apical ectodermal ridge, which soon regresses. The mesenchyme undergoes the process of condensation to form anlagens of prospective skeletal elements. These condensations are surrounded by vascular plexuses. During the course of rudimentation, some mesenchymal cells die, while the others are incorporated into the body wall. Nerve ingrowth into rudimentary limb buds was also detected. The temporary presence of hind limb rudiments in cetacean embryos can be regarded as a good example of recapitulation of phylogenesis in ontogenesis.

Role of Staphylococcus aureus surface adhesins in orthopaedic device infections: are results model-dependent?

Bacterial colonisation of prosthetic material can lead to clinical infection or implant failure, or both, often requiring removal of the device. Adherence of Staphylococcus aureus to bioprosthetic materials is mediated by adhesins belonging to the MSCRAMM (microbial surface components recognising adhesive matrix molecules) family of microbial cell surface proteins. The objective of this study was to compare the virulence of a mutant strain of S. aureus Newman that possesses all three fibrinogen-, fibronectin- and collagen-binding MSCRAMMs (MSCRAMM-positive strain) with that of a mutant strain that lacks all three types of MSCRAMMs (MSCRAMM-negative strain) in a rabbit model of orthopaedic device-related infection. After a hole was drilled into the knee joint of each animal, a group of 10 rabbits was inoculated with the MSCRAMM-positive strain and another group of 10 rabbits received the MSCRAMM-negative strain. A stainless steel screw was then placed into the drilled hole. Two weeks later, the rabbits were killed and serum samples, bone tissue and implants were harvested for bacteriological and histopathological evaluation. No significant difference in infection rates was demonstrated between the two groups. The ability to delineate the role of S. aureus surface adhesins in causing orthopaedic device-related infection could be model-dependent.

[Evaluation of the antihypertensive efficacy and safety of using sustained release verapamil in elderly subjects using ambulatory blood pressure monitoring]. / Ocena skutecznosci hipotensyjnej oraz bezpieczenstwa stosowania werapamilu o przedluzonym dzialaniu u osób w wieku podeszlym przy uzyciu ambulatoryjnego monitorowania cisnienia krwi.

OBJECTIVE: Calcium antagonists are commonly used drugs in the treatment of hypertension in elderly patients. One of the drugs of this group is verapamil. Especially its slow release formula is convenient for use in elderly patients (drug is given once daily). The aim of our study was to evaluate the antihypertensive efficacy of 240 mg slow release verapamil given once daily in elderly hypertensives using ambulatory blood pressure monitoring (ABPM). We also evaluated the safety of this slow release formula using trough to peak ratio. DESIGN AND METHODS: Twenty hypertensives of mean age 67.5 +/- 5.3 were enrolled into the study. Our study was performed using double-blind, placebo-controlled cross-over study design. After a 2-week wash-out period 24-hour ABPM was performed for the first time and then twice: after 2 weeks of placebo or 240 verapamil slow-release and after 2 weeks of verapamil or placebo treatment. The readings prior to and after the treatment were taken using the same procedure. Blood pressure measurements were recorded every 15 minutes between 7.00 and 23.00 and every 30 minutes between 23.00 and 7.00. RESULTS: Verapamil treatment statistically significantly reduced (p < 0.05) mean 24 hour systolic blood pressure from 148.88 +/- 7.56 mmHg to 132.24 +/- 6.13 mmHg and diastolic respectively from 96.68 +/- 6.05 to 83.04 +/- 5.40 mmHg. Heart rate decreased from 77.16 +/- 3.84 BPM to 68.88 +/- 4.41 BPM. Placebo-corrected trough to peak ratio was 70% for systolic blood pressure.

Prenatal development of the integument in Delphinidae (Cetacea: Odontoceti).

The prenatal development of epidermis, dermis, and hypodermis was studied in embryos of different age of two delphinid species (Stenella attenuata, Delphinus delphis), using light and transmission electron microscopical methods. The delphinid embryo is covered by a multilayered tissue formed by four different epidermal generations (periderm, stratum intermedium-I, str. intermedium-II, str. spinosum) produced by the str. basale. The first layer appears at about 40-50 mm of body length, the second type (s.i.-I) about 60-160 mm, and the third type (s.i.-II) is present at 160-500 mm. The first spinosal cells are produced at 225-260 mm body length; thenceforth, the epidermis increases continuously in thickness. Epidermal ridge formation begins about 400-mm body length. The development of the dermis is characterized by the early production of thin connective tissue fibers (40-70-mm body length) and simultaneously the cutaneous muscle matures in structure. Vascular development intensifies between embryos of 150-225 mm, and collagen production increases markedly in fetuses of 225-260-mm length. These events are paralleled by an increase in dermal thickness. The first elastic fibers can be recognized in the skin from the abdomen at about 600-mm body length. The development of the hypodermis is marked by very rapid and constantly progressing growth, beginning about 60-mm body length. The first typical fat cells appear in animals of 360-400 mm. Regional differences are obvious for all skin layers with regard to the flippers, where structural maturation proceeds more rapidly than in dorsal or abdominal regions.

Osteomyelitis associated with pressure sores.

BACKGROUND: Pressure sores may be associated with underlying osteomyelitis that is difficult to differentiate clinically from infection or colonization of adjacent soft tissue. Cultures of bone specimens are frequently contaminated with organisms residing in adjacent soft tissue. The three objectives of this cohort study were to (1) determine the accuracy of clinical evaluation for osteomyelitis; (2) evaluate the potential role of quantitative cultures of bone in differentiating osteomyelitis from soft-tissue infection or colonization; and (3) assess the impact of treating osteomyelitis on the outcome of pressure sores. METHODS: Thirty-six patients with pressure sores related to spinal cord injury or cerebrovascular accident underwent clinical evaluation for osteomyelitis, followed by percutaneous needle biopsy of bone. Routine semiquantitative and quantitative, aerobic and anaerobic cultures of bone specimens were performed. Pathologic examination of bone tissue was used as the standard criterion for diagnosing osteomyelitis. RESULTS: Six (17%) of 36 patients were diagnosed by pathologic examination as having osteomyelitis. The sensitivity and specificity of clinical evaluation were 33% and 60%, respectively. When positive, quantitative bone cultures yielded a similar number of bacterial isolates and a comparable range of bacterial concentration in patients with osteomyelitis vs those without osteomyelitis. Pressure sores healed in all six patients with osteomyelitis after appropriate therapy. CONCLUSIONS: Clinical evaluation for osteomyelitis is often inaccurate. Pathologic examination of bone tissue is required for definitive diagnosis of osteomyelitis. Quantitative bone cultures do not help differentiate osteomyelitis from infection or colonization of adjacent soft tissue. It is possible that treatment of osteomyelitis may improve the outcome of associated pressure sores.

Temporal changes in myocardial uptake on serial bone scans in two cases of presumed senile cardiac amyloidosis.

Two unusual examples of cardiac uptake in elderly patients who had received serial bone scans, one with a "waxing" and the other with a "waning" pattern of myocardial tracer uptake, are presented. An apparent temporal discordance between echocardiographic and scintigraphic findings is noted. Neither of the two patients had clinical, electrocardiographic, or other laboratory evidence of old or new myocardial infarction. Possible causes for the temporal changes are described. Presumptive diagnoses of senile cardiac amyloidosis were made based on clinical findings, noninvasive imaging, and comparison with a historic cohort at the Veterans Administration Medical Center in Houston.

4-MAPC, a 5 alpha-reductase inhibitor, reduces rat ventral prostate weight, DNA, and prostatein concentrations.

Several compounds, such as 4-MAPC (4-methyl-3-oxo-4-aza-5 alpha-pregnane-20- carboxylate), that inhibit conversion of testosterone (T) to dihydrotestosterone (DHT) by 5 alpha-reductase have been demonstrated to reduce prostate size in rats and dogs. The current studies were undertaken to determine if this effect is due to a reduction in cell number, in epithelial cell synthetic activity, or both. Eight-week-old intact rats were treated daily for 14 days with sesame seed oil, 4-MAPC (10 mg/kg), 4-MAPC + testosterone propionate (TP, 1 mg/kg), or 4-MAPC + TP (3 mg/kg). Rats were killed 24 hours after the last injection. In the animals treated only with 4-MAPC, ventral prostate weight was reduced 37%, but the 14% reduction in total DNA was not significant. The mean intraprostatic concentration of prostatein, a major secretory protein, was reduced 45% (P less than 0.05). The 3 mg/kg dose of TP increased ventral prostate weight, prostatein concentrations, and acid phosphatase activity, even though DNA/ventral prostate was similar to that in control animals. These observations indicate that the reduction in ventral prostate weight in adult rats is due in part to a reduction in cell number, but the primary effect was due to a reduction in synthetic activity, and possibly atrophy of the epithelial cells. Furthermore, TP in pharmacologic doses increased ventral prostate weight and synthetic activity without increasing DNA.

The use of immunohistochemistry in metastatic prostatic adenocarcinoma to the breast.

Since the introduction of hormonal therapy for the treatment of metastatic prostatic adenocarcinoma, there have been 33 reports of metastases of prostate carcinoma to the breast. We report two cases of diethylstilbestrol (DES)-treated patients with metastatic prostate adenocarcinoma who developed breast masses. The lesions had infiltrative patterns simulating primary breast carcinoma. Immunoperoxidase stains, prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP) were positive, identifying these cases as metastatic prostatic carcinoma to the breast. Differentiating primary from secondary tumors in these patients is difficult since there have been 10 reports of primary breast carcinoma occurring in DES-treated patients with prostatic adenocarcinoma. Their differentiation is important to direct appropriate therapy, and PSA and PAP immunoperoxidase stains are important in their correct classification.

Subcutaneous fat necrosis associated with pancreatic islet cell carcinoma.

A 75-year-old man with generalized subcutaneous fat necrosis was found to have an islet cell carcinoma of the pancreas. The histologic diagnosis of islet cell carcinoma was confirmed by electron microscopy, which showed characteristic intracytoplasmic granules. This is the second report of the association of islet cell carcinoma of the pancreas with generalized subcutaneous fat necrosis.

Pathologic findings in the hearts of patients with acquired immunodeficiency syndrome.

To determine the extent of myocardial involvement in acquired immunodeficiency syndrome, we reviewed specimens of cardiac tissue obtained during autopsies of 54 patients with this disease. Forty-nine of the specimens showed subtle microscopic changes, the most common being lymphocytic infiltration (49 cases), unevenness of myocardial fibers (34 cases), interstitial fibrosis (28 cases), and myocardial atrophy (22 cases). Twenty-one of the specimens showed a morphologic abnormality that may be classified as a mild cardiomyopathy. Four had microscopic evidence of lymphocytic myocarditis; clinically, however, such myocarditis was diagnosed in only 1 patient, who died of cardiac failure. In a retrospective review, 7 additional hearts (among the 54) showed mild, nonspecific cardiac changes. The causative mechanism and significance of AIDS-related cardiac involvement remain uncertain; nevertheless, physicians should be aware of such involvement, to avoid overburdening the heart with medications that would further impair the myocardium.

Myocardial fibrosis in the elderly.

The increasing interest in geriatric medicine demands an understanding of aging in human tissues. The changes in the human heart are an important aspect of this understanding because cardiovascular diseases are a leading medical problem in the elderly. The published data about age-related changes in the human myocardium remain incomplete and occasionally are controversial. An ongoing study of the hearts of people aged 80 years and older is being conducted at our institution. Various aspects of morphologic changes in these hearts were studied. In this report, we discuss the presence and extent of myocardial fibrosis, namely, the interstitial type. Two types of fibrosis are recognized: (1) scarring, the fibrotic replacement of lost myocardium, usually of vascular origin; and (2) interstitial, in which a delicate fibrotic net encircles single myocardial fibers. The morphologic findings were correlated with the following clinical findings: hypertension, congestive heart failure, emphysema, cor pulmonale, and coronary artery disease. The results support the hypothesis that interstitial fibrosis develops independently of the above-mentioned clinical conditions and may be considered as a true aging process.

Morphometry of the aging heart.

Myocardial structure was studied in seven geriatric patients (cases), aged 79 to 91, and ten younger adult patients (controls), aged 22 to 52. Myocardial sections of posterior left ventricle (PLV), anterior left ventricle (ALV), and ventricular septum (VS) were studied using computerized morphometry. Each section of heart (PLV, ALV, or VS region) was divided into three zones: subepicardial (epi), midmyocardial (myo), and subendocardial (endo). Interstitial collagen content and nuclear and cell diameters of myocytes were obtained. Large scars were excluded. Student's t-test was used to evaluate differences, which were considered significant at a level of P less than 0.001. No significant zonal or regional variation in collagen content was found in control hearts, where interstitial trichrome-positive tissue comprised 6.61% of the myocardium. By contrast, elderly hearts showed a distinct increase in interstitium in the PLV region (18.3%), as compared with ALV and VS, and as compared with control hearts (P less than 0.001). Relative myocyte hypertrophy with aging was most prominent in a zonal midmyocardial distribution (P less than 0.001). Myocyte nuclei showed shrinking with age in the subepicardial zone (P less than 0.001). These findings indicate there are specific age-related alterations in myocardium, which may relate to functional abnormalities seen in the elderly.

On the origin of the so-called Meckelian ossicles in the nasal skull of odontocetes.

Although Cave (1987) accepts the theory that the Meckelian ossicles originate from the maxilloturbinals, evidence given in his study in fact supports the opinion of Klima and van Bree (1985) that the Meckelian ossicles arise from elements of the nasal floor, solum nasi, of the embryonic nasal capsule, in particular from the lamina transversalis anterior and the cartilago paraseptalis.