Practice of radiation therapy for anal cancer in Austria-a survey on behalf of the Austrian radiation oncology society gastrointestinal tumor group (ÖGRO-GIT).

PURPOSE: We conducted a patterns-of-care survey on chemoradiation for locoregionally confined anal cancer in Austria to evaluate areas of disagreement and to identify possible targets for further standardization. METHODS: An anonymous questionnaire comprising 38 questions was sent to all Austrian radiation oncology departments. Results were analyzed descriptively and compared to two international guidelines. RESULTS: The response rate was 93%. Work-up generally includes DRE, endoscopy, and cross-sectional imaging of chest/abdomen and pelvis. PET-CT is used by 38%. Screening for HIV and biopsies of suspicious lymph nodes are infrequently used. All centers perform IMRT, mainly with daily IGRT. Median doses to the primary are 54.7 Gy (T1-2) and 59.4 Gy (T3-4). Suspicious nodes receive a boost (median dose 54 Gy), while elective nodal areas are mainly treated with 45-50.4 Gy. Target delineation of elective nodal areas seems generally uniform, although disagreement exists regarding inclusion of the common iliac nodes. No agreement was found for OAR-delineation and dose constraints. Concurrent chemotherapy is mitomycin and 5­FU/capecitabine. Supportive care beyond skin care is infrequently offered. Intensive follow-up is performed for at least 5 years. Treatment of T1N0 shows considerable disagreement. CONCLUSION: We found a high rate of agreement between the centers and concordance with major guidelines. PET-CT, routine HIV testing, and biopsies of suspicious LN seem underrepresented. The largest controversy regarding target volumes concerns inclusion of the common iliac nodes. Prescribed doses are generally in line with the recommendations or higher. OAR delineation, dose constraints, supportive care, and treatment of early anal cancer represent areas for further standardization.

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes.

Endothelin-1 (ET-1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET-1 we tested the hypothesis that long-term (24 hrs) exposure to ET-1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro-electrode-arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET-1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti-Cx43 antibodies. ET-1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide (P < 0.05), and increased cell areas (P < 0.05) compared to control. ET-1 also caused a time-dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET-1, and was augmented at 24 hrs, compared to controls (P < 0.01). ET-1 increased total Cx43 protein by approximately 40% (P < 0.05) without affecting non- phosphorylated Cx43 (NP-Cx43) protein expression. Quantitative confocal microscopy showed a approximately 30% decrease in the Cx43 immunofluorescence per field in the ET-1 group (P < 0.05) and a reduced field stain intensity (P < 0.05), compared to controls. ET-1-induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET-1 induced susceptibility to arrhythmogenesis.

Competitive strategies in adult beech and spruce: space-related foliar carbon investment versus carbon gain.

In Central Europe, Fagus sylvatica and Picea abies represent contrasting extremes in foliage type, crown structure and length of growing season. In order to examine the competitive strategies of these two co-occurring species, we tested the following hypotheses: (1) the space occupied by the foliage of sun branches is characterized by greater foliar mass investment compared to shade branches, (2) the carbon (C) gain per unit of occupied space is greater in sun than in shade branches, and (3) annual C and water costs of the foliage for sustaining the occupied space are low, wherever C gain per unit of occupied space is low. These were investigated in a mature forest in Southern Germany. The examination was based on the annual assessment of space-related resource investments and gains of the foliage. The foliated space around branches was regarded as the relevant volume with respect to aboveground resource availability. Occupied crown space per standing foliage mass was higher in shade compared to sun branches of beech, whereas no difference existed in crown volume per foliage mass between sun and shade branches of spruce (hypothesis 1 accepted for beech but rejected for spruce). However, beech occupied more space per foliage mass than spruce. The C gain per occupied crown volume was greater in sun than in shade branches (hypothesis 2 accepted) but did not differ between species. The amount of occupied space per respiratory and transpiratory costs did not differ between species or between sun and shade branches. In beech and spruce, the proportion of foliage investment in the annual C balance of sun and shade branches remained rather stable, whereas respiratory costs distinctly increased in shade foliage. Hence, shade branches were costly structures to occupy space, achieving only low and even negative C balances (rejection of hypothesis 3), which conflicts with the claimed C autonomy of branches. Our findings suggest that competitiveness is determined by the standing foliage mass and the annual branch volume increment rather than annual investments in foliage. Expressing competitiveness in terms of space-related resource investments versus returns, as demonstrated here, has the potential of promoting mechanistic understanding of plant-plant interactions.

Comparison of ozone uptake and sensitivity between a phytotron study with young beech and a field experiment with adult beech (Fagus sylvatica).

Chamber experiments on juvenile trees have resulted in severe injury and accelerated loss of leaves along with reduced biomass production under chronically enhanced O3 levels. In contrast, the few studies conducted on adult forest trees in the field have reported low O3 sensitivity. In the present study, young beech in phytotrons was more sensitive to O3 than adult beech in the field, although employed O3 regimes were similar. The hypotheses tested were that: (1) differences in O3 uptake were caused by the ontogenetically higher stomatal conductance of young compared to adult trees, (2) the experimental settings in the phytotrons enhanced O3 uptake compared to field conditions, and (3) a low detoxification capacity contributes to the higher O3 sensitivity of the young trees. The higher O3 sensitivity of juvenile beech in the phytotrons is demonstrated to relate to both the experimental conditions and the physiological responsiveness inherent to tree age.

Spontaneous differentiating primary chondrocytic tissue culture: a model for endochondral ossification.

Primary cartilage-derived cell cultures tend to undergo dedifferentiation, acquire fibroblastic features, and lose most of the characteristics of mature chondrocytes. This phenomenon is due mainly to the close matrix-cell interrelationship typical of cartilage tissue, which is vital for the preservation of the cartilaginous features. In this study we present a model for spontaneous redifferentiation of primary chondrocytic culture. Mandibular condyles excised from 3-day-old mice, thoroughly cleaned of all soft tissue, were digested with 0.1% collagenase. These mandibular condyle-derived chondrocytes (MCDC) were cultured under chondrogenesis-supporting conditions; that is, 5 x 10(5) cells/mL were incubated in Dulbecco's modified Eagle medium supplemented with 100 microg/mL ascorbic acid, 1 mmol/L calcium chloride, 10 mmol/L beta-glycerophosphate, 10% fetal calf serum, and antibiotics. Development and growth rates of these cartilage-derived cultures were determined by following morphological and functional changes. MCDC proliferated intensively during the first 24-48 h following plating, showing fibroblast-like (long spindle-shaped) morphology and producing mainly type I collagen. The proliferation rate gradually declined, and the cells developed polygonal shapes and started to produce type II collagen. In the 10-14-day-old cultures, cells began to aggregate in cartilaginous nodules and exhibited positive staining for acidic Alcian blue, type X collagen, and von Kossa. Expression of core-binding factor alpha(1) increased between 3 and 5 days and declined gradually thereafter. The condylar-derived tissue culture presented here depicts a spontaneous redifferentiation chondrocytic tissue culture that exhibits features of mature chondrocytes typically found in skeletal growth centers. The present study offers a model for primary chondrocytic tissue culture, which might serve as a model for in vitro endochondral ossification.

Photosynthetic capacity in relation to nitrogen in the canopy of a Quercus robur, Fraxinus angustifolia and Tilia cordata flood plain forest.

We measured gas exchange and various leaf parameters of ash (Fraxinus angustifolia Vahl.) and oak (Quercus robur L.) in the high canopy and of lime (Tilia cordata Mill.) in the lower canopy of a planted, 120-year-old floodplain forest in southern Moravia, Czech Republic. The high-canopy leaves of F. angustifolia and Q. robur had nitrogen concentrations on a leaf area basis (N(area)) that were twice those of low-canopy leaves of T. cordata. Upper-canopy leaves of F. angustifolia had a photosynthetic rate at light saturation (A(max)) of about 16 micromol CO2 m(-2) s(-1), whereas A(max) of the upper-canopy foliage of Q. robur achieved only about two thirds of this value. Contrary to previous investigations of photosynthetic performance in monospecific stands, leaves of the uppermost branches of T. cordata at 15-m height had the highest A(max) and transpiration rate among the species studied. Water-use efficiency (WUE) was low in T. cordata at 15-m canopy height, whereas WUE was significantly higher for Q. robur leaves at 27-m height than for the other species. Leaves of T. cordata at 15-m height showed the strongest relationship between A(max) and N(area) (R2 = 0.90) followed by F. angustifolia (R2 = 0.69). The strong correlation between photosynthesis and nitrogen concentration in T. cordata at 15 m, together with the steep regression slope for the A(max):N(area) relationship, indicated that nitrogen allocation to the photosynthetic apparatus resulted in high nitrogen-use efficiency of light-saturated photosynthesis (PNUE). Despite differences in PNUE among species, PNUE was fairly constant for leaves sampled from the same canopy position, suggesting that single-leaf parameters are matched to optimize PNUE for prevailing light conditions. High PNUE in T. cordata at 15 m partially compensated for the species' subordinate position in the canopy, and may be an important mechanism for its coexistence in highly structured vegetation.

Activation of macrophage tumoricidal activity by photodynamic treatment in vitro--indirect activation of macrophages by photodynamically killed tumor cells.

Macrophages constitute a major part of natural tumor defense by their capacity to destroy selectively a broad range of tumor types upon specific activation. In the last couple of years, these cells have also been implicated as effector cells in the destruction of tumors by photodynamic therapy. In the present work, the potential role of macrophage-mediated tumor cytotoxicity after photodynamic treatment in vitro has been investigated with respect to photodynamic activation of macrophages for tumoricidal effector functions. Our data show that photodynamic treatment of highly pure murine bone-marrow-derived macrophages with the hematoporphyrin derivative Photosan-3 does not result in activation of these cells for cytotoxicity against YAC-1 tumor cells or secretion of tumor necrosis factor and nitric oxide, irrespective of co-stimulation with interferon-gamma, a potent priming agent for macrophage antitumoral activity. On the contrary, treatment with higher photosensitizer doses is found to reduce markedly the viability of the macrophage effector cells. Thus, these results do not lend any support to the hypothesis of direct macrophage activation by photodynamic treatment. However, macrophages are found to be activated for tumoricidal effector functions indirectly by photodynamically killed tumor cells, in a way reminiscent of phagocytosis-inducing stimuli. It is thus suggested that recognition and phagocytosis of photodynamically destroyed tumor cells constitutes the major signal for local activation of macrophages in photodynamically treated tumor tissues, which may be crucial for final, specific eradication by the immune system of tumor cells surviving photodynamic treatment.

Cutting edge: differential effect of apoptotic versus necrotic tumor cells on macrophage antitumor activities.

Macrophages (Mphi) play essential roles both in tumor defense and normal tissue homeostasis by removal of transformed as well as damaged and disintegrating cells. Whereas tissue necrosis is known to provoke inflammatory responses, removal of apoptotic cells has been assumed to be immunologically inert. We now show that while Mphi exposure to necrotized tumor cells causes pronounced stimulation of Mphi antitumor activity, exposure of Mphi to apoptotic tumor cells in contrast results in impairment of Mphi-mediated tumor defense and even support of tumor cell growth. Given the fact that apoptosis is a consequence of various cancer treatment modalities, this may lead to a suppression of local antitumor reactions and thus actually counteract endogenous immune-mediated tumor defense mechanisms.

Apoptosis, Fas and Fas-ligand expression in melanocytic tumors.

Impaired regulation of apoptosis is known to be associated with the development of various forms of cancer. Fas binding to its ligand, Fas ligand (Fas-L), has been shown to trigger apoptosis in various cell types. Fas-L is expressed by melanoma cells and has been suggested to play a role in melanoma escape from immune surveillance. In the present study, we assessed apoptotic activity and examined Fas and Fas-L expression in malignant melanomas, Spitz nevi and ordinary melanocytic nevi. We evaluated apoptotic activity using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling. Apoptotic activity was found to be minimal in melanomas and moderate in Spitz nevi. In contrast, common nevi demonstrated significant levels of apoptosis in the deep parts of the tumor. Fas was found to be expressed by all Spitz nevi, most melanocytic nevi and approximately half of the malignant melanoma specimens. Fas expression was also significantly more pronounced in Spitz nevus cells as compared with the two other tumors. The anti-Fas-L antibody was found to stain all three melanocytic tumors. Staining was shown to be stronger and more frequent in melanoma cells as compared to the nevus cells. Using the Spearman test, no significant correlation between Fas-L expression in melanoma cells and apoptosis in MM-infiltrating mononuclear cells was found, suggesting that Fas-L expression in melanoma cells may not be instrumental in their ability to escape immune mechanisms of defense. In contrast, increased levels of apoptosis in the deep parts of melanocytic nevi may reflect and possibly contribute to their benign nature.

Reduced folate carrier (RFC-1) gene expression in normal and psoriatic skin.

Methotrexate is widely used in the treatment of severe psoriasis. However, little is currently known about the mechanisms underlying its therapeutic activity in the skin. Methotrexate has been shown to be carried into cells through the reduced folate carrier (RFC-1). The recent cloning and characterization of the human gene encoding this transmembranal carrier enabled us to investigate RFC-1 gene expression in human skin. Biopsies were obtained from the skin of healthy and psoriatic volunteers. RNA extracted from these biopsies was analyzed by the reverse transcriptase-polymerase chain reaction technique. While RFC-1 gene expression was barely detectable in the uninvolved skin of psoriatic patients and in the skin of healthy volunteers, high levels of RFC-1 transcripts were found in biopsies obtained from psoriatic plaques. To further investigate this pattern of gene expression, we studied skin biopsies by in situ hybridization with a labeled antisense riboprobe specific for the RFC-1 gene. The RFC-1 gene was found to be weakly expressed in the epidermis, in biopsies obtained from the skin of healthy subjects as well as in those from the uninvolved skin of psoriatic patients. In contrast, in biopsies obtained from psoriatic plaques, high levels of RFC-1 gene transcripts were found mostly in the spinous layer of the epidermis. These results suggest the existence of a specific methotrexate carrier in the human epidermis, and may bear relevance to the cutaneous manifestations of methotrexate toxicity.

Effect of photodynamic pretreatment on the susceptibility of murine tumor cells to macrophage antitumor mechanisms.

In vitro photodynamic treatment of YAC-1 murine T-lymphoma cells with the hematoporphyrin derivative Photosan 3 and red light resulted in dose-dependent phototoxicity. Photodynamic pretreatment, however, did not render these cells more susceptible to macrophage-mediated tumor cytotoxicity or the cytotoxic effects of macrophage-derived antitumor mediators like tumor necrosis factor alpha (TNF-alpha) or interferon beta (IFN-beta). Independent of the degree of photosensitization used, the cytotoxicity values obtained with macrophages or the different mediators were shifted by the respective values for phototoxicity, suggesting these effects to be additive and thus not interdependent. These data show that while higher overall tumor cytotoxicity can be achieved by a combination of photodynamic treatment and macrophage-mediated tumor destruction, this apparently is not a result of enhanced sensitivity of photodynamically treated tumor cells to macrophage antitumor mechanisms in general.

Localized granuloma annulare. Histopathological and direct immunofluorescence study of early lesions, and the adjacent normal-looking skin of actively spreading lesions.

We studied 10 biopsies of early (< 1 week) localized granuloma annulare (LGA) lesions and 10 specimens of the normal-looking skin adjacent to actively spreading LGA lesions for signs of vasculitis, using histological and direct immunofluorescent (DIF) techniques. In the early LGA lesions, the collagen fibers showed various forms of alterations, with hyalinization and fragmentation being the most common; in half of these lesions, neutrophils and nuclear fragments in various numbers and densities were found among the altered collagen fibers. Some of the small blood vessels in the areas of granulomatous inflammation showed endothelial cell hypertrophy in four cases, and in one case also endothelial cell proliferation and luminal occlusion. In only one case, a single small blood vessel, which was situated in the center of a palisading granuloma, showed fibrinoid necrosis of its walls. The DIF study of all 20 specimens and the histological study of the 10 specimens of normal-looking adjacent skin did not reveal immune deposits in the vessels' walls, or histological evidence of vasculitis, respectively. We believe that these findings do not support a role of vasculitis in the formation of LGA lesions, but suggest that neutrophils might play a primary or secondary role in their pathogenesis.

The long-term followup of primary anterior cruciate ligament repair. Defining a rationale for augmentation.

Fifty primary ACL repairs using the Marshall multiple suture technique were analyzed. The average age at surgery was 23 years (range, 15 to 56), with 76% under the age of 30. The average followup was 61.3 months (range, 48 to 86). The average time from injury to surgery was 7 days (range, 1 to 18). Eighty percent of the injuries were sports-related with football and skiing predominating. Thirty-eight percent were "isolated" ACL tears, and 62% had associated injuries. There was a 46% incidence of meniscal tear with 59% of the meniscal tears being repaired. The postoperative evaluation included a multifactorial analysis correlating 43 variables including subjective, objective, radiographic, and KT-1000 data. The Hospital for Special Surgery Knee Diagnostic Score, a clinical objective score based on the postoperative Lachman and pivot shift examination, a KT-1000 arthrometer data score, and an overall combined assessment score were determined. The results showed 59% excellent, 18% good, 14% fair, and 8% poor. The Lachman test was diagnostic in all cases. The quality of ACL tissue at repair was rated excellent or good in 62% of the cases. Four patterns of ACL tears were distinguished by the location of the tear. Football injury, younger age, increased peroperative pivot shift, midsubstance Type IV tear, and return of full motion correlated with poor postoperative results. Increasing age, tight jointedness, Type I tears, and a 5 degrees flexion contracture correlated with good postoperative results.

Rationale and protocol for postoperative anterior cruciate ligament rehabilitation.

A rehabilitation program for postoperative anterior cruciate ligament (ACL) reconstruction permits adequate tissue healing time and allows early protected muscular conditioning. The program is based on kinematic, biomechanical, and kinesiologic factors as they pertain to ACL function. The program is divided into five phases. The early phases, zero to 12 weeks, are intended to control translational forces across the ACL and to allow necessary time for ligament revascularization and soft-tissue healing. During this period, isometric and isotonic training of the hamstrings and quadriceps muscle groups in a restricted active arc of motion is permitted. Passive motion is encouraged, and progressive weight bearing is allowed. Conditioning of the upper body as well as the nonsurgical lower limb is stressed. During the advanced phases, 12-31 weeks, isotonic muscle training continues throughout a full range of motion. Greater strength, coordination, and endurance to achieve dynamic stability of the knee, preparing the patient for unrestricted activity, are stressed. Approximately 52 weeks of active rehabilitation after ACL reconstruction surgery are usually required to return to a preinjury level of function.

Application of a direct agglutination test for detection of specific anti-Leishmania antibodies in the canine reservoir.

A direct agglutination test (DAT) for detection of visceral leishmaniasis in humans has been developed. In this study, it was evaluated for applicability to detection of infections in dogs, a reservoir species. The reliability of the test was improved by treating the test sera with 0.2 M 2-mercaptoethanol and incubating them at 37 degrees C. Sensitivity was 100% and specificity was 98.9% when the test was used on serum samples from 220 dogs, including 26 with parasitologically confirmed canine leishmaniasis, 12 with suspected but unconfirmed leishmaniasis, and 182 with other conditions. The DAT detected specific antibodies in 10 dogs with canine leishmaniasis diagnosed by case history, clinical signs of leishmaniasis, and seropositivity in an immunofluorescence test using either promastigotes or amastigotes, as well as in 2 dogs suspected of having leishmaniasis. The performance of an antigen prepared from a homologous isolate of Leishmania infantum in the DAT was compared with that of an antigen from a laboratory-adapted strain of L. donovani (sensu lato). The homologous antigen compared favorably with the standard antigen, and the results provided further evidence of the potential of the DAT for detection of Leishmania infection in the canine reservoir host. The results of this study, together with those of our previous studies in human visceral leishmaniasis, demonstrate that the DAT is highly suitable for wide-scale epidemiological and ecological field work. This technique could also facilitate diagnosis of leishmaniasis in dogs in veterinary health services.

Recently developed methods for the detection of babesial infections.

During the acute phase of babesiosis the direct identification of the causative agent is possible by examination of stained blood smears. An improved identification procedure for the diagnosis of Babesia bovis using DNA hybridization has been reported recently. For the diagnosis of both acute and chronic babesiosis, various serodiagnostic techniques have been developed. Recent advances in serodiagnosis designed for the detection of anti-Babesia antibodies were partly achieved by improving and standardizing the indirect immunofluorescence and complement fixation tests. The use of soluble antigens derived from Babesia culture supernatants or enriched, purified merozoites in the micro-enzyme-linked immunosorbent assay, solid phase radioimmunoassay and latex agglutination test has provided increased sensitivity and specificity in detecting anti-Babesia antibodies. Immunoblots and monoclonal antibodies have been used for the characterization and identification of babesial antigens and antigenic determinants. The advantages and disadvantages as well as the area of application of the currently available test systems will be discussed.

[Imported leishmaniasis of dogs: clinical aspects, diagnosis and therapy with Na stibogluconate (Pentostam)]. / Die importierte Hundeleishmaniose: Erfahrungen zu Klinik, Diagnostik und Therapie mit Na-Stibogluconat (Pentostam).

Between May 1982 and September 1986 13 dogs with leishmaniosis received inpatient treatment with Pentostam at the I. Medical Department of the Veterinary Faculty, University of Munich. Predominant clinical signs of all animals were alterations of the skin and hair coat and additionally they showed a reduced general condition (4 dogs), loss of weight (3 dogs), diarrhea (5 dogs) and pain during movements. In the blood samples taken prior treatment in 9 dogs there was an increased total protein concentration. 5 dogs had changes in the red and 4 dogs in the white blood count. Thrombocytopenia occurred in 6 dogs. Leishmaniosis was diagnosed clinically and serologically (IFAT). Na-Stibogluconate (Pentostam) was used for the treatment at a dosage of 10 or 20 mg/kg on 10 or 14 consecutive days. Two dogs died during an early stage of therapy. 6 dogs were discharged as clinically cured after a single or repeated treatment course(s).