Portable dynamic ultrasonography is a useful tool for the evaluation of suspected syndesmotic instability: a cadaveric study.

PURPOSE: Portable ultrasonography (P-US) is increasingly used to diagnose syndesmotic instability. The aim of this study was to evaluate syndesmotic instability by measuring the distal tibiofibular clear space (TFCS) in a cadaveric model using P-US with progressive stages of syndesmotic ligamentous transection under external rotation stress. METHODS: Ten fresh lower leg cadaveric specimens amputated above the proximal tibiofibular joint were used. Using P-US, the TFCS was evaluated in the intact stage and after progressive sectioning of the (1) anterior-inferior tibiofibular ligament (AITFL), (2) interosseous ligament (IOL), and (3) posterior-inferior tibiofibular ligament (PITFL). The TFCS was measured in both the unstressed (0 Nm) state and with 4.5, 6.0, 7.5, and 9.0 Nm of external rotation stress using a bone hook placed on the first metatarsal bone at each stage of ligamentous transection stage using both P-US and fluoroscopy. RESULTS: When assessed with P-US, partial syndesmotic injury encompassing the AITFL and IOL resulted in significant TFCS widening at 4.5 Nm of external rotation torque when compared to intact state with a TFCS-opening of 2.6 ± 2 mm, p = 0.01. In contrast, no significant differences in TFCS were detected using fluoroscopy. Only a moderate correlation was found between P-US and fluoroscopy. CONCLUSION: P-US is a useful tool in diagnosing syndesmotic instability during external rotation stress examination. TFCS-opening increased as additional ligaments of the syndesmosis were transected, and application of 4.5 Nm torque was sufficient to detect a difference of 2.6 mm after the IOL cut.

Revision of the Massarineae (Pleosporales, Dothideomycetes).

We here taxonomically revise the suborder Massarineae (Pleosporales, Dothideomycetes, Ascomycota). Sequences of SSU and LSU nrDNA and the translation elongation factor 1-alpha gene (tef1) are newly obtained from 106 Massarineae taxa that are phylogenetically analysed along with published sequences of 131 taxa in this suborder retrieved from GenBank. We recognise 12 families and five unknown lineages in the Massarineae. Among the nine families previously known, the monophyletic status of the Dictyosporiaceae, Didymosphaeriaceae, Latoruaceae, Macrodiplodiopsidaceae, Massarinaceae, Morosphaeriaceae, and Trematosphaeriaceae was strongly supported with bootstrap support values above 96 %, while the clades of the Bambusicolaceae and the Lentitheciaceae are moderately supported. Two new families, Parabambusicolaceae and Sulcatisporaceae, are proposed. The Parabambusicolaceae is erected to accommodate Aquastroma and Parabambusicola genera nova, as well as two unnamed Monodictys species. The Parabambusicolaceae is characterised by depressed globose to hemispherical ascomata with or without surrounding stromatic tissue, and multi-septate, clavate to fusiform, hyaline ascospores. The Sulcatisporaceae is established for Magnicamarosporium and Sulcatispora genera nova and Neobambusicola. The Sulcatisporaceae is characterised by subglobose ascomata with a short ostiolar neck, trabeculate pseudoparaphyses, clavate asci, broadly fusiform ascospores, and ellipsoid to subglobose conidia with or without striate ornamentation. The genus Periconia and its relatives are segregated from the Massarinaceae and placed in a resurrected family, the Periconiaceae. We have summarised the morphological and ecological features, and clarified the accepted members of each family. Ten new genera, 22 new species, and seven new combinations are described and illustrated. The complete ITS sequences of nrDNA are also provided for all new taxa for use as barcode markers.

A giant gamma-ray flare from the magnetar SGR 1806-20.

Two classes of rotating neutron stars-soft gamma-ray repeaters (SGRs) and anomalous X-ray pulsars-are magnetars, whose X-ray emission is powered by a very strong magnetic field (B approximately 10(15) G). SGRs occasionally become 'active', producing many short X-ray bursts. Extremely rarely, an SGR emits a giant flare with a total energy about a thousand times higher than in a typical burst. Here we report that SGR 1806-20 emitted a giant flare on 27 December 2004. The total (isotropic) flare energy is 2 x 10(46) erg, which is about a hundred times higher than the other two previously observed giant flares. The energy release probably occurred during a catastrophic reconfiguration of the neutron star's magnetic field. If the event had occurred at a larger distance, but within 40 megaparsecs, it would have resembled a short, hard gamma-ray burst, suggesting that flares from extragalactic SGRs may form a subclass of such bursts.

Analysis of Chinese rabies virus isolates from 2003-2007 based on P and M protein genes.

UNLABELLED: Phylogenetic analysis of nucleotide sequences or deduced amino acid sequences of phosphoprotein (P protein), matrix (M) protein, and glycoprotein (G protein) genes of 18 Chinese isolates of Rabies virus (RABV) from 2003-2007 showed that these isolates formed a separate monophyletic lineage consisting of sub-lineages A and B. Compared with laboratory-fixed strains, recent Chinese isolates of sub-lineage B contained Val or Ala instead of Met69 in P protein, which is involved in generating truncated P proteins. In addition, one of these isolates CHpg3 had Pro instead of Ser63 and Leu instead of Ser64. Importantly, all functional domains of P and M proteins of all recent Chinese isolates were similar to those of laboratory-fixed strains. This study showed that although the recent Chinese RABV isolates belonged to a distinct lineage, their functional domains of P and M proteins were highly conserved. KEYWORDS: rabies virus; glycoprotein; phosphoprotein; matrix protein; China.

Thermography for the detection of Secondary Raynaud's Phenomenon by means of the Distal-Dorsal Distance.

Raynaud's phenomenon (RP) is a disease characterized by a transient ischemic process, in an exaggerated vascular response to cold or emotional stress. Thermography is a resource applied to support diagnosis of changes in the circulatory system. The aim of the study was to use the DistalDorsal Thermography Difference (DDD) in thermographic images to assess thermal behavior in individuals with secondary RP. The research was carried out in the period between 2018 and 2019. The sample means of the Distal-consisted of 44 individuals in a control group (Control) and 44 individuals in a pathological group (RP2). The participants, after acclimatization, were submitted to the cold stress protocol. The protocol consisted of immersing hands in a container of water at a temperature of 15°C for 60 seconds. The acquisition of thermographic images was performed at the pre-test moment and at the 1st, 3rd, 5th, 7th, 10th and 15th minute. At each time, the DDD values (of all fingers - minimum, maximum and sum) between the groups were analyzed. For statistical analysis, the independent t test and Cohen's d test were used. Regarding the results, there was a difference in relation to the rate of temperature recovery between the groups. The first group showed a rate of reheating just after the first minute subsequent to the cold stress test, while the RP2 group was unable to recover the temperature over 15 minutes. DDD, regardless of the selected criterion, proved to be a valid index for verifying the temperature gradient in the study with individuals identified with secondary RP.

Continuous culture of rat C6 glioma in serum-free medium.

In this communication we describe serum-free culture conditions for the serial propagation of the C6 glioma cell line. The growth rate, saturation density, and morphology of these cells are equivalent to those of their serum-grown counterparts when cultured in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's medium F-12 supplemented with trace elements, insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and a serum-spreading factor (SSF) partially purified from human plasma. The requirement for SSF in the medium can be satisfied by preincubating the tissue culture dishes with SSF. Tissue culture dishes sequentially pretreated with poly-D-lysine and purified cold insouluble globulin will also substitute for this requirement. The fatty acid-free bovine serum albumin/linoleic acid complex increases the growth rate of these cells but has no appreciable effect on their morphology, saturation density, or ability to grow with repeated subculture. The growth stimulation caused by this complex appears to be dependent on the fatty acid, as the fatty acid-free bovine serum albumin alone has no effect on the growth rate. Linoleic acid is cytotoxic in the absence of bovine serum albumin, and the fatty acid-free bovine serum albumin prevents this toxicity. Other fatty acids including oleic, arachidonic, and palmitic only partially substitute for the growth-promoting effect of linoleic acid.

Improved in vitro survival of normal, functional spleen cells.

An enrichment method for improving the viability of monolayers of normal mouse spleen cells is described. These cells are capable of saving lethally x-irradiated recipient mice from death, presumably by proliferating and differentiating into mature blood cells.

Establishment of four functional, clonal strains of animal cells in culture.

The single-cell plating technique was used to develop four clonal cell lines that perform organ-specific functions after being serially cultured for prolonged periods. These strains include steroid-secreting Leydig cells, melanomacells that form pigment, and two strains from a hormone-secreting rat pituitary tumor. One of the cell lines from the pituitary tumor secretes growt hormone, while another line derived from the same tumor secretes a substance similar to adrenocorticotropic hormone.

Differentiated rat glial cell strain in tissue culture.

Rat glial tumors, induced by injections of N-nitrosomethylurea, were plated and propagated in culture. Among a few cell strains obtained, one clone contains S-100 protein, which is unique to brain in vertebrates. Stationary-phase cultures contain approximately ten times more S-100 protein per cell than exponentially growing cells. When injected into newborn rats, cells producing S-100 grew as a glial tumor, which contained S-100 protein.

Genetic and phylogenetic characterization of rabies virus isolates from wildlife and livestock in Paraiba, Brazil.

Thirty-four rabies virus (RV) isolates from foxes (8), insectivore bats (9), cattle (14), sheep (1), a goat (1) and a donkey (1) from Paraiba state, northeastern Brazil, were genetically characterized. Sequences of 890 nts of nucleoprotein (N) genes of these isolates were analyzed and compared with those of other Brazilian isolates characterized earlier. Phylogenetic analysis revealed three genetical lineages of RV co-existing in this region. Each lineage was found to be associated with particular host species and to circulate independently of each other. The first lineage was found in foxes (Dusicyon sp.) and could be discriminated from domestic carnivore isolates from Sao Paulo, Goias and Minas Gerais in the southern and central Brazil. The second lineage was associated with insectivorous bats (Molossus spp.) and differed from vampire bat-associated RV isolates. The third lineage was found in livestock and clustered with vampire bat-associated RV isolates from Sao Paulo, Tocantins, Goias and Matto Grosso. These results indicate that RV of these genetic lineages are cocirculating in the Paraiba state and that livestock in this region are infected with vampire bat-associated RV, suggesting that the vampire bat is the main reservoir of livestock rabies in this region.

Growth characteristics of primary tissue cultures from heterotransplanted human colorectal carcinomas in serum-free medium.

Procedures are described for the preparation of reproducible primary cultures from human colorectal tumors transplanted in the athymic nude mouse and for the quantitative evaluation of growth by means of counting suspensions of nuclei from these cultures with a Coulter counter. Growth curves of primary cultures from 11 colorectal tumors in serum-free medium are shown and discussed with respect to the in vitro conditions to be met for the propagation of in vivo stem cell populations.

Relationship of cell growth behavior in vitro to tumorigenicity in athymic nude mice.

The serum requirements, anchorage requirements, saturation densities, and contact inhibition responses of a variety of mammalian cell lines were determined under uniform conditions. The serum requirement of both transformed and normal cells was a sensitive function of initial plating density. Cloning efficiency on irradiated mouse monolayers was found to be an invalid indicator of contact inhibition of growth, since most cell lines that failed to form visible colonies on cell monolayers nonetheless proliferated on these monolayers. When normal and neoplastic cells from a variety of sources were examined, none of the growth parameters that serve to define the transformed state in vitro correlated consistently with cellular tumorigenicity in athymic nude mice. It is concluded that the most reliable and physiologically meaningful assay for malignant transformation is, at present, cellular tumorigenicity in athymic nude mice.

Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies.

Monoclonal antibodies (MoAbs) were raised against epidermal growth factor (EGF) receptors on a human epidermoid carcinoma cell line, A431. Administration of anti-EGF receptor MoAbs inhibited tumor formation in athymic mice by A431 cells and by another epidermal carcinoma cell line, T222. When one of the same MoAbs was used in therapy against Li-7 (a human hepatoma) and HeLa cells (a cervical carcinoma), tumor growth was not affected. The number of EGF receptors on A431 cells was about 100-fold higher than on T222, Li-7, and HeLa cells, suggesting that the number of EGF receptors may not be an important determinant in suppressing tumor growth. Three anti-EGF receptor MoAbs were used in the present studies. MoAbs 528 (immunoglobulin G2a) and 225 (immunoglobulin G1) are capable of competing with EGF for receptor binding and inhibit proliferation of A431 cells in culture. The other MoAb, 455 (immunoglobulin G1), is incapable of blocking the binding of EGF to its receptors and has no effect on the proliferation of cultured A431 cells. All three MoAbs inhibited A431 tumor growth in athymic mice, indicating that the antibody isotype and the site of binding on the EGF receptor are not the determinants of antiproliferative activity in vivo. The observation that MoAb against the receptor for EGF is cytostatic rather than cytocidal in vitro against A431 cells, yet completely prevents tumor growth in vivo, suggests that some host animal responses also may be involved in the antitumor effect. MoAbs against growth factor receptors could provide useful immunotherapeutic agents.

Growth control of heterologous tissue culture cells in the congenitally athymic nude mouse.

A variety of heterologous mammalian cells were inoculated into nude mice and scored for tumorigenicity. The cells tested were from primary cell cultures, established cell lines of neoplastic origin, established cell lines of nontumor origin, and primary cell cultures transformed by oncogenic viruses. Regardless of the animal species of origin, every cell line that was tumorigenic in some other animal host and every cell line of neoplastic origin was tumorigenic in nude mice. Several tissue culture cells lines capable of indefinite growth in vitro failed to form tumors in nude mice, and the basis of this growth suppression was investigated. The findings suggest that the failure of an established cell line to form tumors in nude mice is an authentic response to host-mediated growth-regulatory signals.

Prognostic value of nucleolar protein p120 in patients with resected lung adenocarcinoma.

PURPOSE: In this study we investigated the prognostic significance of proliferation-associated nucleolar protein p120 in primary resected lung adenocarcinoma because it reflects tumor growth fractions in vitro. PATIENTS AND METHODS: Expression levels of p120 in tumors were assessed by immunohistochemistry in 74 patients who underwent radical resection. With clinical follow-up data, the prognostic significance of p120 calculated by labeling indices was evaluated using the Cox proportional hazards model. RESULTS: p120 protein was clearly detected in nucleoli of adenocarcinoma cells. Its expression levels widely varied in each sample from 8.5% to 67. 2%, with a mean +/- SD of 35.2% +/- 15.1%. No significant correlation was found between expression levels of p120 and clinicopathologic factors. However, the expression levels of p120 were negatively correlated with the tumor doubling time calculated with retrospective chest roentgenograms. Using a cutoff value of 35% in the labeling index of p120, patients with high expression of p120 experienced early recurrence and shorter survival compared with those who had low expression of p120. Multivariate analysis showed that p120 served as an independent, as well as the strongest, prognostic factor for resected lung adenocarcinoma. CONCLUSION: This report provides the first evidence that expression levels of p120 in tumor tissues can be used as an independent and powerful prognostic marker for resected lung adenocarcinoma.

Hormones and factors that stimulate growth of a rat islet tumor cell line in serum-free medium.

A number of hormones and factors were found to stimulate growth of cultured rat islet tumor cells, the RIN-r cell line. A serum-free supplemented medium from RIN-r cells was formulated. It consisted of a 1:1 mixture of Ham's F12 and DME media with the addition of insulin, transferrin, triiodothyronine, prolactin, growth hormone, and an extract of proteose peptone (medium IM). The growth rate of RIN-r cells in this medium is as great as it is in 10% serum-supplemented medium. Up to 10 populations doublings occurred over a period of 20 days. Insulin is a very effective mitogen for RIN-r cells and has an effect on concentrations as low as 30 ng/ml. In addition, the insulin-like somatomedin, multiplication stimulating activity (MSA), is a growth factor at 50 ng/ml. It was found that RIN-r cells proliferate and continue to produce immunoreactive insulin in a hormonally and nutritionally defined medium. This medium is derived from medium IM, in which insulin is replaced with MSA and proteose peptone is omitted. Variations of this medium may prove useful in studies on the growth and function of the normal islets in long-term primary culture.

Secretion pattern of pro-opiomelanocortin-derived peptides by a pituitary adenoma from a patient with Cushing's disease.

Fragments of the pituitary adenoma of a patient with Cushing's disease were maintained in defined culture medium. Immunoreactive (IR) ACTH, IR alpha-MSH, IR beta-lipotropin (beta-LPH), IR beta-endorphin, and IR gamma-MSHs secreted from the adenoma were studied with gel permeation chromatography and the respective RIAs. The adenoma secreted roughly equimolar quantities of IR beta-LPH plus IR beta-endorphin, IR gamma 3-MSHs, and IR ACTHs. It also secreted IR alpha-MSH as well as IR gamma 1-MSH, although in a much lower concentration than the above four peptides. The secreted gamma 3-MSH-like peptides were found to be glycosylated. The secretion pattern suggests that this particular adenoma processes the pro-opiomelanocortin molecule in pathways which encompass those of both the pars distalis and the pars intermedia.

GABA and benzodiazepine receptors in the gerbil brain after transient ischemia: demonstration by quantitative receptor autoradiography.

Quantitative receptor autoradiography was used to measure the binding of gamma-aminobutyric acid (GABA) and benzodiazepine receptors after ischemia by means of transient occlusion of bilateral common carotid arteries in the gerbil. [3H]Muscimol was used to label the GABAA receptors and [3H]flunitrazepam to label central type benzodiazepine receptors. In the superolateral convexities of the frontal cortices, [3H]muscimol binding was increased in 60% of the animals killed 3 days after ischemia, and decreased in 67% of the animals killed 27 days after ischemia. Twenty-seven days after ischemia, [3H]flunitrazepam binding in the substantia nigra pars reticulata increased to 252% of the control, though the increase in [3H]muscimol binding was not significant. In the dorsolateral region of the caudate putamen, marked neuronal necrosis and depletion of both [3H]muscimol and [3H]flunitrazepam binding sites were observed 27 days after ischemia, the ventromedial region being left intact. In spite of the depletion of pyramidal cells in the CA1 region of the hippocampus, both [3H]muscimol and [3H]flunitrazepam binding sites were preserved 27 days after ischemia. Since our previous study revealed that adenosine A1 binding sites were depleted in the CA1 subfield of the hippocampus after ischemia correlating with neuronal damage, GABAA and benzodiazepine receptors may not be distributed predominantly on the pyramidal cells in the CA1 region.

Phylogenetic relationships between adenoviruses as inferred from nucleotide sequences of inverted terminal repeats.

The nucleotide (nt) sequences of inverted terminal repeats (ITR) from human adenovirus (Ad) 19, bovine Ad1 (BAd1), bovine Ad3 (BAd3), canine Ad2 (CAd2) and an avian Ad, EDS-76, were determined. The length of the ITR sequence was 160 bp in Ad19, 159 bp in BAd1, 195 bp in BAd3, 196 bp in CAd2 and 52 bp in EDS-76. CAd2 had the longest ITR among the examined Ads, BAd3 the second longest, and EDS-76 had the shortest ITR. A TAAT sequence located between the 10th and 13th nt counted from the ends was conserved in all Ads examined so far. To determine phylogenetic relationships among human and animal Ads, sequences of their ITRs were compared, and a phylogenetic tree was constructed by using the maximum-likelihood method. It is the method involving statistical analysis of computing the probability of a particular set of sequences on a given tree and maximizing this probability over all evolutionary trees [Felsenstein, J. Mol. Evol. 17 (1981) 368-376]. From these analyses, it was found that members belonging to the same human Ad subgenus are related closely to each other, whereas representatives of different human subgenera are distributed rather divergently among animal Ads.

Induction of antitumor immunity by combined immunogene therapy using IL-2 and IL-12 in low antigenic Lewis lung carcinoma.

Interleukin-2 (IL-2) and interleukin-12 (IL-12) are crucial cytokines that induce potent antitumor responses in a variety of animal cancer models. Although single gene transfer of either IL-2 or IL-12 exhibits limited antitumor effects, the combination of IL-2 and IL-12 has been shown to induce a stronger antitumor response and to cure tumor-bearing mice. To examine the conditions necessary for tumor rejection, we varied the local concentration of IL-2 and IL-12 by introducing these genes into Lewis lung carcinoma (LLC) cells via retroviral vectors and/or an adenoviral vector and evaluated the growth of inoculated LLC cells (5 x 10(5) cells). In contrast to the result when using a stepwise dose increase of IL-2 either without or with a fixed production of IL-12 (4-5 ng/5 x 10(5) cells/24 hours, insufficient for tumor rejection by itself), rejection of the tumor was achieved in 75% of the mice when the IL-12 secretion was combined with high and transient IL-2 production (42 ng/5 x 10(5) cells/24 hours) using additional adenoviral vector transduction (100 multiplicities of infection). An abundant infiltration of both CD4+ (47.4/mm2) and CD8+ (85.6/mm2) T cells was a characteristic finding in the dual gene-transfected LLC tumors. Importantly, consistent with the rejection of rechallenged parental cells, tumor-specific cytotoxic T lymphocytes were induced only from the splenocytes of mice inoculated with the dual gene-transduced LLC cells, suggesting the existence of protective antitumor memory. In addition, only vaccination of dual gene-transduced LLC cells inhibited the growth of pre-established LLC tumors. These results indicate that generation of a pivotal antitumor response likely depends on the synergistic combination and concentration of IL-2 and IL-12 in the local milieu by which tumor-specific immune memory is established.