Cryopreservation of redwood (Sequoia sempervirens) in vitro buds using vitrification-based techniques.

In this study, the efficiency of three vitrification-based cryopreservation techniques, i.e. vitrification, encapsulation-vitrification and droplet-vitrification were compared for cryopreserving Sequoia sempervirens apical and basal buds sampled from in vitro shoot cultures. The effect of cold-hardening of mother-plants and of bud culture medium and sucrose preculture was also investigated. Culture of apical and basal buds sampled from cold-hardened mother-plants on Quoirin and Lepoivre medium with activated charcoal had a positive effect on regrowth. Only droplet-vitrification ensured survival and regrowth after cryopreservation. After cryopreservation, regeneration of apical buds was possible for PVS2 exposure durations between 90 and 180 min but it remained low, with a maximum of 18 percent after 135 min treatment. With basal buds, regeneration after cryopreservation was possible over a larger range of PVS2 treatment durations, between 30 and 180 min. The highest regeneration percentage was slightly higher (22 percent) than that measured with apical buds, and was also achieved after 135 min PVS2 exposure.

The role of external fixation in the management of infected avascular necrosis after traumatic talus fractures.

PURPOSE: Avascular necrosis (AVN) after fractures of the talus is a distinct and challenging clinical entity that is associated with poor outcomes. Although several articles are published on the management of posttraumatic AVN of the talus, very little is known about the management of infected AVN after talus fractures. Therefore, three cases of infected AVN were treated successfully by extensive debridement, external fixation and arthrodesis. METHODS: Three cases of infected AVN of the talus were encountered after a mean of 3 months (range 2-6 months) after initial reconstructive surgery. Suspected infection was confirmed by positron emission tomography scan (PET-CT). Management involved extensive debridement, PMMA cement if necessary and final fusion using medial external fixator, accompanied by culture guided antibiotics. Functional outcome was assessed using the Foot Function Index (FFI) and the American Orthopaedic Foot and Ankle Society hindfoot score (AOFAS). Quality of life (QOL) was measured by the EuroQol-5D (EQ-5D). RESULTS: After a mean follow up of 24 months (range 13-29), FFI index scores ranged from poor to good (23, 50, 56) with similar AOFAS scores indicating poor to fair functional outcome (38, 41, 71). The EQ-5D score was 0.78. Overall patient satisfaction was high with a mean VAS of 8.3 (range 8-9). CONCLUSION: Infected talar AVN is a rare condition associated with severe long-term morbidity in term of joint function. The authors recommend extensive debridement and arthrodesis by means of external fixation, followed by post-operative culture-guided antibiotics for the treatment of infected avascular necrosis of traumatic talar fractures. Shared decision-making and expectation management are of crucial importance and may lead to high patient satisfaction despite low functional outcomes. LEVEL OF EVIDENCE: IV, Retrospective case series.

Systematic review: Diagnostics, management and outcome of fractures of the posterior process of the talus.

BACKGROUND: Fractures of the posterior process of the talus are rare and frequently overlooked, possibly leading to pseudo-arthrosis and chronic pain. To gain more insight into the diagnosis, treatment and outcome of fractures of the posterior process of the talus (PPTF), a systematic review of the current literature was performed to provide recommendations for the management of PPTF. METHODS: A literature search in the electronic databases of PubMed, EMbase, Google Scholar and Cochrane library was performed in January 2020 to identify all clinical studies on PPTF with more than three patients. Amongst other variables, the type of study, number of patients, mechanism of injury, type of fracture (anatomy), imaging modality, treatment, postoperative protocol, outcomes, complications and duration of follow-up were noted for systematic analysis of the available evidence, adherent to the PRISMA guidelines. RESULTS: Seven original studies were included with a total of 66 patients. More than one third of patients presented with a (sub)talar joint dislocation (n = 25, 37.9%) and 51.5% sustained associated ipsilateral lower extremity fractures (n = 34). Delayed diagnosis occurred in 36.4% of patients (n = 24). Out of 48 patients with outcome data available, 41.7% (n = 20) reported impaired function. In the non-operative group, 64.7% (n = 11) had impaired functional outcome, compared to 33.3% (n = 6) in the ORIF group, and 30.8% (n = 4) in the fragment excision group (p < 0.001). One third of the patients developed one or more complications (n = 25, 37.9%), mostly found in the non-operatively treated group (73.7%, n = 14) compared to ORIF (25.0%, n = 8, p < 0.001). CONCLUSION: Early recognition and timely treatment is warranted in order to achieve pre-injury functional outcome and reduce morbidity. Given the significantly higher complication rate and lower return to the previous level of functionality reported after non-operative treatment, ORIF is recommended if there is (even minimal) displacement, articular involvement or if the fracture extends into the talus body.

Patients at high risk for CMV infection and disease show delayed CD8+ T-cell immune recovery after allogeneic stem cell transplantation.

Human cytomegalovirus (CMV) is a major cause of death after transplantation. The frequency of pp65-specific T cells was examined in 38 HLA-A2+ stem cell recipients during the first year after transplantation. Patients were divided into four groups based on donor/recipient serostatus: d+/r+ (n=17), d+/r- (n=7), d-/r+ (n=9) and d-/r- (n=5). Peripheral blood mononuclear cells were stimulated with the CMVpp65 peptide NLVPMVATV, and the specific T-cell frequency was assessed by interferon gamma (IFN-gamma) ELISPOT assay. Responding T cells were characterized by flow cytometry revealing a terminal differentiated effector phenotype. Surveillance of CMV infection was carried out by real-time polymerase chain reaction (n=26) or immunofluorescence (n=12). Infection was present in 7/9 d-/r+ high-risk patients, and CMV disease occurred exclusively in this group with delayed or absent virus-specific T-cell recovery. In contrast, 16/24 intermediate-risk patients showed CMV-specific T cells. Our data suggest that CMV infection and disease rates are elevated in high-risk patients with delayed CMV-specific T-cell immune reconstitution and lower in those with early recovery of T-cell immunity. We recommend preferring CMV seropositive donors for CMV seropositive recipients, as this should lead to durable CMV-specific T-cell responses soon after transplantation with consecutive protection from CMV disease.

Gunshot induced injuries in orthopaedic trauma research. A bibliometric analysis of the most influential literature.

A growing burden of gunshot injuries demands evidence-based ballistic trauma management. No comprehensive systematic overview of the current knowledge is available to date. This study aims to identify and analyze the most influential publications in the field of orthopedic ballistic trauma research. All databases available in the Thomson Reuters Web of Knowledge were searched to conduct this bibliometrical study. The most cited orthopedic ballistic trauma articles published between 1950 and 2015 were identified by use of a multi-step approach. Publications with ten citations and more were analyzed for citations, journal, authorship, geographic origin, area of research, anatomical site, study type, study category, and level of evidence. Citations of the 128 included studies ranged from 113 to 10. These were published in fifty different journals between 1953 and 2011. Most publications (n=106; 83%) originated from the USA, were retrospective (n=85; 66.4%), level IV studies (n=90; 70.3%), reported on spinal gunshot injuries (n=49; 38.33%) and were published between 1980 and 2000 (n=111; 86.7%). This bibliometric study provides the first comprehensive overview of influential publications in the field of orthopedic ballistic trauma research. More prospective studies and high-quality systematic reviews are needed. Centres with a high burden of gunshot injuries from the developing world need to share their experience in form of international publications, to provide a more comprehensive picture of the global gun-related orthopedic injury burden. TYPE OF STUDY: bibliometric analysis: level III.

CD spectroscopy provides evidence for excitonic interactions involving red-shifted chlorophyll forms in photosystem I.

Selective destruction of the strongly dichroic red-shifted chlorophyll form (C709 nm) in photosystem I (PSI) trimers from Spirulina, by either non-selective high intensity illumination (photobleaching) or incubation with low concentrations of Triton X-100 is accompanied by changes in the circular dichroism spectrum of the same amplitude and of opposite sign at 677 nm. The data are interpreted in terms of a dimeric chlorophyll structure with excitonic bands at these two wavelengths. Similar photobleaching experiments with PSI-200 from maize also suggest the presence of bulk antenna/red form excitonic interactions.

Autochthonous hepatitis E virus infection in Germany with sequence similarities to other European isolates.

Hepatitis E virus (HEV) is a common cause of acute hepatitis in endemic areas. Yet reports on autochthonous cases in other areas such as middle Europe are increasing. Here we report on a patient, who obviously acquired his HEV infection in Germany. Sequence analysis of the virus gained from his serum revealed homologies to other European isolates and swine isolates.

HLA type-independent method to monitor polyoma BK virus-specific CD4 and CD8 T-cell immunity.

(Re)activation of quiescent viral diseases is a major problem in immunosuppressed transplant patients. Polyoma BK virus-associated nephropathy (PVAN) caused by active polyoma BK virus (BKV) infection became a main reason for graft loss in kidney transplantation. After diagnosis, most transplant centers react by reducing immunosuppression (IS) to allow the immune system to control the infection. However, the impact of reduced IS on BKV immunity is not well researched. Here we present an HLA type-independent method to monitor BKV-specific T-cell immunity. Applying our method, viral protein 1-specific CD4+ and CD8+ T-cell responses were detected in patients with serum BKV-DNA levels >250 000 copies/mL. In addition, specific T-cell responses were also found in allograft-infiltrating cells. The method can be used to assess the impact of decreased immunosuppression on BKV immunity and to clarify the role of specific T cells in the pathogenesis of PVAN. We strongly recommend its implementation in future clinical studies.

[Quantitative and morphological studies of retinal development in mice with trisomy 19]. / Quantitative und morphologische Untersuchungen der Retinaentwicklung bei Trisomie-19-Mäusen.

To investigate the development of the retinal layers, the eyes of mice with trisomy 19 have been examined by light microscopy between the 2nd and 15th postnatal day. The diameter of the eye, thickness of the entire retina and both relative thickness and nuclear density of each of the retinal layers have been measured and compared to those of chromosomally balanced control animals. Malformations of the eye, alterations of cell morphology or disturbed lamination can not be observed. Retinal differentiation of trisomy 19 mice is delayed by approximately two days. The development of all cellular constituents, i.e., of both neuroectodermal and mesenchymal origin, is retarded accordingly. The eyes of trisomy 19 mice are of reduced size. The relative thickness of each retinal layer follows a normal growth pattern; there is no indication for a selective impairment of the development of one particular layer. With the exception of the ganglion cell layer, nuclear densities of each retinal layer do not differ from those of control mice. The comparison of nuclear densities in the ganglion cell layer suggests that in trisomy 19 mice fewer postmitotic cells differentiate into mature retinal cells.

[Correlation between resistance testing and typing of Candida albicans--isolates from AIDS patients and chronic recurrent oral candidiasis]. / Korrelation zwischen Resistenztestung und Typisierung von Candida albicans--Isolaten bei AIDS-Patienten und chronisch rezidivierender oraler Candidose.

In patients with AIDS and recurrent oropharyngeal candidosis results of biotyping and serotyping were compared with antifungal susceptibility testing. Biotype B1 was found predominantly with 44.3% from 169 C. albicans isolates. Serotype A was seen in 150/169 C. albicans isolates. No close correlation between high MIC values (> or = 25 micrograms/ml) for C. albicans isolates against fluconazole nor a change to a particular biotype or serotype has been observed in recurrences of oral candidosis.

Comparative evaluation of three antifungal susceptibility test methods for Candida albicans isolates and correlation with response to fluconazole therapy.

In vitro susceptibilities were determined for 56 Candida albicans isolates obtained from the oral cavities of 41 patients with human immunodeficiency virus infection. The agents tested included fluconazole, itraconazole, ketoconazole, flucytosine, and amphotericin B. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P (M27-P micro), a broth microdilution technique using high-resolution medium (HR micro), and the Etest with solidified yeast-nitrogen base agar. The in vitro findings were correlated with in vivo response to fluconazole therapy for oropharyngeal candidiasis. For all C. albicans isolates from patients with oropharyngeal candidiasis not responding to fluconazole MICs were found to be > or = 6.25 micrograms/ml by the M27-P micro method and > or = 25 micrograms/ml by the HR micro method as well as the Etest. However, for several C. albicans isolates from patients who responded to fluconazole therapy MICs found to be above the suggested breakpoints of resistance. The appropriate rank order of best agreement between the M27-P micro method and HR micro method was amphotericin B > fluconazole > flucytosine > ketoconazole > itraconazole. The appropriate rank order with best agreement between the M27-P micro method and the Etest was flucytosine > amphotericin B > fluconazole > ketoconazole > or = itraconazole. It could be concluded that a good correlation between in vitro resistance and clinical failure was found with all methods. However, the test methods used in this study did not necessarily predict clinical response to therapy with fluconazole.

Correlation between antifungal susceptibility testing of Candida isolates from patients with HIV infection and clinical results after treatment with fluconazole.

In an open-label controlled study 23 HIV-infected patients (CDC IV A-E) with documented oropharyngeal candidosis were treated with 100 mg fluconazole orally over 5 days (53 episodes; 1-6 treatments/patient). Efficacy data were compared with a control group of 21 patients who received treatment for 10-21 days with 100 mg fluconazole for candidosis. Candida isolates were repeatedly recovered from patients before and after treatment with fluconazole and antifungal susceptibility testing (microbroth-dilution) was done. Inoculum size, medium pH, incubation time and temperature were standardized. Up to 85% of patients responded to therapy clinically and mycologically. Candida albicans was the most important yeast (86%) isolated from cultures of oral washings. In 90% of C. albicans isolates MIC to fluconazole were low (< or = 1.56 mg/l). Primary resistance to fluconazole was not seen, but secondary resistance occurred in two cases clinically and in vitro (MIC > or = 25 mg/l). Short treatment for 5 days was as successful as for 10 to 21 days without leading to significantly more recurrences of oral candidosis in these patients. Selection of Candida spp. other than C. albicans (e.g. Candida krusei, Torulopsis glabrata) under repeated fluconazole treatment occurred rarely. One patient developed clinical signs of chronic recurrent candidiasis, where only C. krusei could be cultured repeatedly.

Emergence of fluconazole-resistant strains of Candida albicans in patients with recurrent oropharyngeal candidosis and human immunodeficiency virus infection.

After repeated use of fluconazole for therapy of oropharyngeal candidosis, the emergence of in vitro fluconazole-resistant Candida albicans isolates (MIC, > or = 25 micrograms/ml) together with oral candidosis unresponsive to oral dosages of up to 400 mg of fluconazole were observed in patients with human immunodeficiency virus (HIV) infection. Antifungal susceptibility testing was done by broth microdilution and agar dilution techniques on C. albicans isolates recovered from a cohort of patients with symptomatic HIV infection who were treated repeatedly with fluconazole for oropharyngeal candidosis. In vitro findings did show a gradual increase in the MICs for C. albicans isolates recovered from selected patients with repeated episodes of oropharyngeal candidosis. Primary resistance of C. albicans to fluconazole was not seen. Cross-resistance in vitro occurred between fluconazole and other azoles (ketoconazole, itraconazole), but to a lesser extent. The results of the study suggest that the development of clinical resistance to fluconazole could be clearly correlated to in vitro resistance to fluconazole. Itraconazole may still serve as an effective antifungal agent in patients with HIV infection and oropharyngeal candidosis nonresponsive to fluconazole.

Femoropopliteal bypass with a compliant, composite polyurethane/Dacron graft: short-term results of a multicentre trial.

A new, compliant, highly porous, non-woven, polyurethane vascular prosthesis has been developed in an effort to improve on the performance of currently available prosthetic grafts for infrainguinal reconstruction. From April 1990 to August 1991, 57 femoropopliteal bypass grafts were implanted in 47 patients by surgeons at five university centres. In all instances, the saphenous vein was unavailable, unusable or reserved for use elsewhere. An empirical perioperative risk score for acute occlusion (0-20) was developed, based on such factors as severity of clinical ischaemia, quality of inflow and outflow, site of distal anastomosis and associated drug therapy. Primary cumulative patency was calculated according to standard life-table analysis. Poor inflow and a distal anastomosis below the knee were significant factors affecting graft patency (P = 0.001 and P = 0.001 respectively). Six-month cumulative patency for the 25 grafts with good inflow and above or mid-knee anastomoses (79%) was superior to the cumulative patency for all 57 grafts (59%). There was a significant improvement in patency rates between 'low' (22 grafts) and 'medium risk' (27 grafts) patients and 'high risk' (eight grafts) ones (risk scores 0-10 and 11-20 respectively) at a level of P = 0.001. There were two operative deaths (4%). Of the 19 postoperative occlusions, six occurred within 30 days and 18 within 6 months. These data indicate that the patency rates achieved with this new graft compare favourably with other available prosthetic grafts. In addition, the graft demonstrates superior handling characteristics and eliminates bleeding through suture holes.(ABSTRACT TRUNCATED AT 250 WORDS)

A new acid-fast trichrome stain for simultaneous detection of Cryptosporidium parvum and microsporidial species in stool specimens.

The detection in stool specimens of Cryptosporidium parvum and microsporidia, the most frequent parasitic pathogens causing diarrhea in AIDS patients, until now has depended on two different staining methods. However, since double infections occur and minimization of laboratory costs is mandatory, development of a method for simultaneous detection of these parasites appeared desirable. We report on a new, inexpensive, and easy-to-perform staining procedure to demonstrate both acid-fast oocysts of C. parvum and other coccidia, as well as microsporidial spores. This acid-fast trichrome stain yields results comparable to those obtained by the Kinyoun and modified trichrome methods and considerably reduces the time necessary for microscopic examination.

Thermal behavior of long wavelength absorption transitions in Spirulina platensis photosystem I trimers.

In photosystem I trimers of Spirulina platensis a major long wavelength transition is irreversibly bleached by illumination with high-intensity white light. The photobleaching hole, identified by both absorption and circular dichroism spectroscopies, is interpreted as the inhomogeneously broadened Q(y) transition of a chlorophyll form that absorbs maximally near 709 nm at room temperature. Analysis of the mean square deviation of the photobleaching hole between 80 and 300 K, in the linear electron-phonon frame, indicates that the optical reorganization energy is 52 cm(-1), four times greater than that for the bulk, short-wavelength-absorbing chlorophylls, and the inhomogenous site distribution bandwidth is close to 150 cm(-1). The room temperature bandwidth, close to 18.5 nm, is dominated by thermal (homogeneous) broadening. Photobleaching induces correlated circular dichroism changes, of opposite sign, at 709 and 670 nm, which suggests that the long wavelength transition may be a low energy excitonic band, in agreement with its high reorganization energy. Clear identification of the 709-nm spectral form was used in developing a Gaussian description of the long wavelength absorption tail by analyzing the changing band shape during photobleaching using a global decomposition procedure. Additional absorption states near 720, 733, and 743 nm were thus identified. The lowest energy state at 743 nm is present in substoichiometric levels at room temperature and its presence was confirmed by fluorescence spectroscopy. This state displays an unusual increase in intensity upon lowering the temperature, which is successfully described by assuming the presence of low-lying, thermally populated states.