Depletion of CLL-associated patrolling monocytes and macrophages controls disease development and repairs immune dysfunction in vivo.

Chronic lymphocytic leukemia (CLL) is characterized by apoptosis resistance and a dysfunctional immune system. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in vivo. Using the Eµ-TCL1 mouse model, we observed severe skewing of myeloid cell populations with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes also accumulated in the spleen in a CCR2-dependent manner, and were severely skewed toward Ly6C(low) patrolling or nonclassical phenotype. In addition, the percentage of MHC-II(hi) dendritic cells and macrophages significantly dropped in the spleen. Gene expression profiling of CLL-associated monocytes revealed aberrantly high PD-L1 expression and secretion of multiple inflammatory and immunosuppressive cytokines like interleukin-10, tumor necrosis factor-α and CXCL9. In vivo myeloid cell depletion using liposomal Clodronate resulted in a significant control of CLL development accompanied by a pronounced repair of innate immune cell phenotypes and a partial resolution of systemic inflammation. In addition, CLL-associated skewing of T cells toward antigen-experienced phenotypes was repaired. The presented data suggest that targeting nonmalignant myeloid cells might serve as a novel immunotherapeutical strategy for CLL.

[Operative treatment of displaced intra-articular calcaneal fractures]. / Osteosynthese dislozierter intraartikulärer Kalkaneusfrakturen.

OBJECTIVE: Anatomic reduction of displaced intra-articular calcaneal fractures with restoration of height, length, and axial alignment and reconstruction of the subtalar and calcaneocuboid joints. INDICATIONS: Displaced intra-articular calcaneal fractures with incongruity of the posterior facet of the subtalar joint, loss of height, and axial malalignment. CONTRAINDICATIONS: High perioperative risk, soft tissue infection, advanced peripheral arterial disease (stage III), neurogenic osteoarthropathy, poor patient compliance (e. g., substance abuse). SURGICAL TECHNIQUE: Extended lateral approach with the patient placed on the uninjured side. Reduction of the anatomic shape and joint surfaces according to the preoperative CT-based planning. Reduction of the medial wall and step-wise reconstruction of the posterior facet from medial to lateral. Reduction of the tuberosity and anterior process fragments to the posterior joint block and temporary fixation with Kirschner wires. Internal fixation with an anatomic lateral plate in a locking or nonlocking mode. Alternatively less invasive internal fixation with a calcaneus nail over a sinus tarsi approach for less severe fracture types. POSTOPERATIVE MANAGEMENT: The lower leg is immobilized in a brace until the wound is healed. Range of motion exercises of the ankle and subtalar joints are initiated on the second postoperative day. Patients are mobilized in their own shoe with partial weight bearing of 20 kg for 6-12 weeks depending on fracture severity and bone quality. RESULTS: Over a 4-year period, 163 patients with 184 displaced, intra-articular calcaneal fractures were treated with a lateral plate via an extended approach. In all, 102 patients with 116 fractures were followed for a mean of 8 years. A surgical revision was necessary in 4 cases (3.4%) of postoperative hematoma, 2 (1.7%) superficial and 5 (4.3%) deep infections. Of the latter, 2 patients needed a free flap for definite wound coverage, no calcanectomy or amputation was needed. Secondary subtalar fusion for symptomatic posttraumatic arthritis was performed in 9 cases (7.8%). At follow-up, the AOFAS Ankle/Hindfoot Score averaged 70.2, the Zwipp Score averaged 76.0, the German versions of the Foot Function Index and SF-36 physical component averaged 32.8 and 42.2, respectively. Scores were significantly lower with increasing fracture severity according to the Sanders and Zwipp classifications, bilateral fractures, open fractures, and with work-related injuries. With less invasive fixation using a calcaneal nail, superficial wound edge necrosis was seen in 2 of 75 cases (2.7%).

Fractures of the sustentaculum tali.

OBJECTIVE: Anatomical reconstruction of displaced sustentaculum tali fractures via a direct medial approach. INDICATIONS: Displaced fractures of the sustentaculum tali with incongruity or depression of the medial facet of the subtalar joint, entrapment of the flexor hallucis longus or flexor digitorum longus tendons, fracture line extending into the posterior facet of the subtalar joint. CONTRAINDICATIONS: Infected or grossly contaminated soft tissue, severely restricted vascular supply to the foot, high perioperative risk. SURGICAL TECHNIQUE: Direct medial approach over the sustentaculum tali, retraction of the tendons, joint exploration, fracture reduction using the medial facet and cortical outline as guidelines, fracture fixation with two small fragment screws from medial to lateral directed slightly plantarly and posteriorly. Fractures with depression of the medial facet as a whole can alternatively be reduced and fixed percutaneously. POSTOPERATIVE MANAGEMENT: Lower leg splint for 5-7 days, partial weight-bearing with 20 kg for 6-8 weeks (until radiographic signs of consolidation) in the patient's own shoewear, early range of motion exercises of the ankle, subtalar and mid-tarsal joints. RESULTS: Over a course of 15 years, 31 patients were treated operatively for sustentacular fractures. In all, 27 patients (87%) had additional fractures to the same foot and ankle. Eighteen patients with a mean age of 41 years treated at our institution with screw fixation for a unilateral fracture of the sustentaculum tali could be followed for a mean of 80 months (range 15-151 months). No wound healing problems or infections were seen with the medial approach. At the time of follow-up, 15 sustentaculum tali fractures had an average Foot Function Index of 21.6 and an average AOFAS Ankle-Hindfoot Score of 83.6. Patients with isolated fractures of the sustentaculum tali had significantly better scores than those with additional injuries. In 1 patient, an additional lateral process fracture of the talus required subtalar fusion due to persistent pain. Care must be taken not to overlook these atypical calcaneal fractures and accompanying injuries to the mid-tarsal joint and the lateral talar process as seen in 45% and 23%, respectively, in the present series.

[Minimally invasive fixation of calcaneal fractures]. / Minimal-invasive osteosynthese von kalkaneusfrakturen.

OBJECTIVE: Anatomic reduction of displaced calcaneal fractures with minimal soft tissue alteration. INDICATIONS: Extra-articular and selected intra-articular calcaneal fractures (simple fracture pattern: Sanders type II, critical soft tissue conditions, contraindications to open reduction), temporary stabilization of complex injuries or polytraumatized patients. CONTRAINDICATIONS: Impossible percutaneous reduction and fixation. SURGICAL TECHNIQUE: Gross reduction of the main fragments is achieved with a Schanz screw introduced percutaneously into the tuberosity fragment. Fine reduction is obtained through percutaneous manipulation of the fragments wit Kirschner wires, Steinmann pins, sharp and smooth elevators via stab incisions. Anatomic reduction of the subtalar joint is controlled arthroscopically in cases of displaced intra-articular fractures. Fixation is achieved with screws introduced percutaneously. POSTOPERATIVE MANAGEMENT: Early range of motion exercises of the ankle and subtalar joints are initiated the first postoperative day. Beginning on postoperative day 2, patients are mobilized with partial weight bearing for 6-8 weeks. As soon as the edema has subsided, patients are encouraged to wear their own shoes. RESULTS: Between 1998 and 2008, 68 patients were treated with definite percutaneous fixation for displaced calcaneal fractures. In 37 patients with intraarticular fractures (Sanders types IIA and IIB), anatomic joint reduction was verified with subtalar arthroscopy. No soft tissue-related complications were observed. Thirty-five patients were followed for a minimum of 2 years postoperatively, the average was 5 years postoperatively. Subjectively, 33 of 35 patients were satisfied with the clinical outcome. The AOFAS Hindfoot Score averaged 90.7 (range 64-100) at a mean of 5 years after surgery. Percutaneous screw fixation of calcaneal fractures is associated with minimal soft tissue traumatization and low complication rates. It allows early rehabilitation and excellent results with proper patient selection. With intra-articular fractures, proper reduction of the articular surface has to be confirmed intraoperatively.

[Allergies to animals and fungi]. / Allergien auf Tiere und Pilze.

Allergies to animals are behind the house-dust mite allergy the most frequent cause for indoor allergic respiratory symptoms. In case of persistent allergen exposure symptoms like rhinitis, itch of the skin or asthma are usually not perceived intensively and, thus, can not assigned to an animal or an animal source. In many cases animal allergies are based on a perennial allergen exposure. Although most likely all animals may be the cause of a respiratory allergy, cats, dogs, and horses are the most frequent elicitors. The diagnosis of an allergy to an animal needs to be set with due care, since it often causes emotional reactions, diverse conflicts, but also lack of understanding. Rarer are allergies to fungi even though fungi as allergen sources since decades belong to the differential diagnosis in respiratory allergies particularly in case of late summer asthma. Fungi are ubiquitous and present indoors as well as outdoors. Unfortunately the field of fungal allergy is not well explored and diagnostic possibilities are limited. The most promising therapy in both allergy to animals and fungi would be complete avoiding of contact with the respective allergen source. Indeed many preventive recommendations are given; however, realization is often not successful. In selected cases specific immunotherapy for both animal and fungal allergies is a potential therapeutic option.

Simulating the impact of genetic diversity of Medicago truncatula on germination and emergence using a crop emergence model for ideotype breeding.

BACKGROUND AND AIMS: Germination and heterotrophic growth are crucial steps for stand establishment. Numerical experiments based on the modelling of these early stages in relation to major environmental factors at sowing were used as a powerful tool to browse the effects of the genetic diversity of Medicago truncatula, one of the model legume species, under a range of agronomic scenarios, and to highlight the most important plant parameters for emergence. To this end, the emergence of several genotypes of M. truncatula was simulated under a range of sowing conditions with a germination and emergence simulation model. METHODS: After testing the predictive quality of the model by comparing simulations to field observations of several genotypes of M. truncatula, numerical experiments were performed under a wide range of environmental conditions (sowing dates × years × seedbed structure). Germination and emergence was simulated for a set of five genotypes previously parameterized and for two virtual genotypes engineered to maximize the potential effects of genetic diversity. KEY RESULTS: The simulation results gave an average value of 5-10 % difference in final emergence between genotypes, which was low, but the analysis underlined considerable inter-annual variation. The effects of parameters describing germination and emergence processes were quantified and ranked according to their contribution to the variation in emergence. Seedling non-emergence was mainly related to mechanical obstacles (40-50 %). More generally, plant parameters that accelerated the emergence time course significantly contributed to limiting the risk of soil surface crusting occurring before seedling emergence. CONCLUSIONS: The model-assisted analysis of the effects of genetic diversity demonstrated its usefulness in helping to identify the parameters which have most influence that could be improved by breeding programmes. These results should also enable a deeper analysis of the genetic determinism of the main plant parameters influencing emergence, using the genomic tools available for this model plant.

Using a model-based framework for analysing genetic diversity during germination and heterotrophic growth of Medicago truncatula.

BACKGROUND AND AIMS: The framework provided by an emergence model was used: (1) for phenotyping germination and heterotrophic growth of Medicago truncatula in relation to two major environmental factors, temperature and water potential; and (2) to evaluate the extent of genetic differences in emergence-model parameters. METHODS: Eight cultivars and natural accessions of M. trunculata were studied. Germination was recorded from 5 to 30 degrees C and from 0 to -0.75 MPa, and seedling growth from 10 to 20 degrees C. KEY RESULTS: Thermal time to reach 50 % germination was very short (15 degrees Cd) and almost stable between genotypes, while base temperature (2-3 degrees C) and base water potential for germination (-0.7 to -1.3 MPa) varied between genotypes. Only 35 degrees Cd after germination were required to reach 30 mm hypocotyl length with significant differences among genotypes. Base temperature for elongation varied from 5.5 to 7.5 degrees C. Low temperatures induced a general shortening of the seedling, with some genotypes more responsive than others. No relationship with initial seed mass or seed reserve distribution was observed, which might have explained differences between genotypes and the effects of low temperatures. CONCLUSIONS: The study provides a set of reference values for M. trunculata users. The use of the ecophysiological model allows comparison of these values between such non-crop species and other crops. It has enabled phenotypic variability in response to environmental conditions related to the emergence process to be identified. The model will allow simulation of emergence differences between genotypes in a range of environments using these parameter values. Genomic tools available for the model species M. trunculata will make it possible to analyse the genetic and molecular determinants of these differences.

[Pansclerotic porphyria cutanea tarda after chronic exposure to organic solvents]. / Pansklerotische Skleroporphyrie nach langjähriger Exposition gegenüber organischen Lösungsmitteln.

A 63 year old man developed generalized scleroderma with massive sclerotic areas, particularly in the abdominal region, four years after being diagnosed with porphyria cutanea tarda (PCT). He had almost daily exposure to organic solvents (benzene, trichlorethylene) for many years. The cutaneous fibrosis progressed dramatically leading to a pansclerosis, even though the uroporphyrin levels were borderline and the liver enzyme values were normal. Organic solvents are among those substances which can cause a cutaneous fibrosis. The unusually complicated clinical development in our patient was a combination of the two initial factors, the PCT and the long term exposure to organic solvents. The pansclerotic PCT was differentiated from a systemic sclerosis, a disabling pansclerotic morphea and a generalized morphea by means of histological examinations, the absence of a Raynaud phenomenon and the non-involvement of additional organs. Auto-antibodies typical for systemic sclerosis were negative. Using a medium dosage of UVA1 phototherapy and intensive physiotherapy, the progression of the skin disease was stopped and the sclerosis improved.

In vitro antioxidant activity of non-cultivated vegetables of ethnic Albanians in southern Italy.

A total of 27 extracts from non-cultivated and weedy vegetables traditionally consumed by ethnic Albanians (Arbëreshë) in the Vulture area (southern Italy) were tested for their free radical scavenging activity (FRSA) in the DPPH (1,1-diphenyl-2-picrylhydrazil radical) screening assay, for their in vitro non-enzymatic inhibition of bovine brain lipid peroxidation and for their inhibition of xanthine oxidase (XO). In both antioxidant assays strong activity was shown for Leopoldia comosa (bulbs, syn.: Muscari comosum) and Centaurea calcitrapa (young whorls). In the lipid peroxidation assay, extracts from leaves of Origanum heracleoticum, Urtica dioica and Tordylium apulum showed a remarkable inhibitory activity (> 50%), too. In the case of Leopoldia comosa and Origanum heracleoticum this activity was comparable to quercetin (at a concentration of 50 microM) and Rhodiola rosea extract. Extracts from non-cultivated Cichorium intybus, Chondrilla juncea and Stellaria media showed strong in vitro inhibition of xanthine oxidase, with an activity higher than that of a reference extract from Ledum groenlandicum. These findings suggest that weedy vegetables may be useful antioxidants of interest in the prevention of ageing related diseases, CNS disorders and as potential sources of phytomedicines against hyperuricaemia and gout.

Cutaneous leukocytoclastic vasculitis in dermatomyositis suggests malignancy.

BACKGROUND: Dermatomyositis (DM) is a rare connective tissue disease which has been shown to be associated with an underlying malignancy. OBJECTIVE: Evaluation of the prevalence of malignancy in DM at our clinic and search for characteristics of the paraneoplastic form of disease. METHODS: Retrospective review of patient files and histology reports over the period from 1991 to 1998. RESULTS: 23 patients (14 women and 9 men) with DM could be identified in this time period with a median age at diagnosis of 48 years. Malignancies were found in 5 (22%) cases. The skin biopsies of all patients showed features of DM; in 7 cases, a leukocytoclastic vasculitis was detected. Four of the 5 cases with an associated malignancy demonstrated histologically a vasculitis in lesional skin, compared to only 3 out of 18 cases without malignancy (p < 0.05, Fisher's exact test). CONCLUSION: Our data suggest that vasculitis in lesional skin biopsies has a predictive value for the presence of underlying malignancy.

Joys and pitfalls of fermi surface mapping in Bi(2)Sr(2)CaCu(2)O(8+delta) using angle resolved photoemission

On the basis of angle-scanned photoemission data recorded using unpolarized radiation, with high (E,k) resolution, and an extremely dense sampling of k space, we resolve the current controversy regarding the normal state Fermi surface (FS) in Bi(2)Sr(2)CaCu(2)O(8+delta). The true picture is simple, self-consistent, and robust: the FS is holelike, with the form of rounded tubes centered on the corners of the Brillouin zone. Two further types of features are also clearly observed: shadow FSs, which are most likely to be due to short range antiferromagnetic spin correlations, and diffraction replicas of the main FS caused by passage of the photoelectrons through the modulated Bi-O planes.

Mechanism of ion permeation in skeletal muscle chloride channels.

Voltage-gated Cl- channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl- channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I- and related anions. Extracellular and intracellular I- exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br-) and polyatomic (SCN-, NO3-, CH3SO3-) anions. In addition, I- block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I- causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I- slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined KD values for I- block in three distinct kinetic states and found that binding of I- to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I- binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.

Monitoring the introduction of laparoscopic cholecystectomy in a district general hospital.

There has been recent concern regarding the widespread introduction of laparoscopic procedures without adequate training. We report a 2 year prospective series of 229 consecutive open and laparoscopic cholecystectomies, following the introduction of laparoscopic surgery at a district general hospital. All laparoscopic cholecystectomies were performed by surgeons without formal training in this procedure. The proportion of laparoscopic cholecystectomies rose from 27% in 1992 to 62% in 1993, with a conversion rate of 14%. An overall complication rate of 23% was recorded, 29% for open cholecystectomy and 16% for laparoscopic cholecystectomy. Procedure specific complication rates were 6% and 3% respectively. No major bile duct injuries occurred and the 30-day mortality was 0.9%. We conclude that laparoscopic cholecystectomy has been introduced as a safe procedure in this hospital, as compared to open cholecystectomy. It is recommended that new techniques should be introduced carefully and monitored by means of prospective audit.

Carbon and electron flow in Clostridium butyricum grown in chemostat culture on glycerol and on glucose.

The metabolism of Clostridium butyricum DSM 5431 was studied in chemostat culture under carbon limitation using either glucose or glycerol. On glycerol, the enzymes glycerol dehydrogenase, diol dehydratase and 1,3-propanediol (1,3-PD) dehydrogenase constitute the branch point that partitions the carbon flux between the competing pathways, i.e. formation of either 1,3-PD or acetate and butyrate. The increasing levels of these enzyme activities with increasing dilution rates (D) explained the constant proportion of glycerol conversion into 1,3-PD. The production of acetate or butyrate constitutes another important branch point and when D increased (i) large amounts of intracellular acetyl-CoA accumulated, (ii) the carbon flux switched from butyric acid to acetic acid, (iii) the specific activity of thiolase was not affected, suggesting this enzyme may be the bottleneck for carbon flux to butyrate biosynthesis providing an explanation for the accumulation of large amounts of intracellular acetyl-CoA, and (iv) high levels of NADH were found in the cell. Oxidation of NADH by 1,3-PD dehydrogenase was linked to the production of 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase. The fact that high intracellular concentrations of NADH were found means that diol dehydratase activity is the rate-limiting step in 1,3-PD formation, avoiding the accumulation of 3-HPA which is a very toxic compound. The specific rate of glucose catabolism (q glucose = 11.1 mmol h-1 g-1) was around four times lower than the specific rate of glycerol catabolism (q glucose = 57.4 mmol h-1 g-1). On glucose-grown cells, reducing equivalents which are released in the glycolytic pathway were reoxidized by the butyric pathway and the low specific formation rate of butyric acid led to an increase in the intracellular level of acetyl-CoA and NADH. Carbon flow was higher on glycerol due to the reoxidation of NADH by both butyric and PD pathways.

Isolation and Characterization of Clostridium butyricum DSM 5431 Mutants with Increased Resistance to 1,3-Propanediol and Altered Production of Acids.

Clostridium butyricum mutants were isolated from the parent strain DSM 5431 after mutagenesis with N-methyl-N(prm1)-nitro-N-nitrosoguanidine and two selection procedures: osmotic pressure and the proton suicide method. Isolated mutants were more resistant to glycerol and to 1,3-propanediol (1,3-PD) than was the wild type, and they produced more biomass. In batch culture on 62 g of glycerol per liter, the wild type produced more acetic acid than butyrate, with an acetate/butyrate ratio of 5.0, whereas the mutants produced almost the same quantities of both acids or more butyrate than acetate with acetate/butyrate ratios from 0.6 to 1.1. The total acid formation was higher in the wild-type strain. Results of analysis of key metabolic enzymatic activities were in accordance with the pattern of fermentation product formation: either the butyrate kinase activity increased or the acetate kinase activity decreased in cell extracts of the mutants. A decreased level of the hydrogenase and NADH-ferredoxin activities concomitant with an increase in ferredoxin-NAD(sup+) reductase activities supports the conclusion that the maximum percentage of NADH available and used for the formation of 1,3-PD was higher for the mutants (97 to 100%) than for the wild type (70%). In fed-batch culture, at the end of the fermentation (72 h for the wild-type strain and 80 to 85 h for the mutants), 44% more glycerol was consumed and 50% more 1,3-PD was produced by the mutants than by the wild-type strain.

Bifidobacteria and probiotic effects: action of Bifidobacterium species on conjugated bile salts.

The effect of six different conjugated bile salts (two trihydroxyconjugated bile salts: tauro and glycocholic acids; and four dihydroxyconjugated bile salts: tauro- and glycochenodeoxycholic, tauro- and glycodeoxycholic acids) on eight bifidobacteria strains were studied. A strong growth-inhibitory effect was observed (80% at 0.95 mM) for each bile salt and strain. This phenomenon was explained by the production of deconjugated bile salt during bifidobacteria growth. The deconjugation phenomenon was concurrent with biomass production, and deconjugated bile salts were the sole compound produced during bifidobacteria biotransformation. In resting cell experiments, differences appeared between the strains and the kind of bile salts, particularly concerning taurocholic acid. The Bifidobacterium longum strains were the most efficient among the bacteria tested.

Detection of two hypervariable (ATTTT)n loci in the human genome.

Object of this investigation was the isolation of a single-locus probe from a multi-locus fingerprint. Individual specific multi-locus fingerprints in man were generated by using the oligonucleotide probe (ATTTT)5. An isolated (ATTTT)5-positive DNA fragment was analyzed using polymerase chain reaction (PCR) cycle-sequencing and nonradioactive direct-blotting electrophoresis. A digoxigenated oligonucleotide synthesized according to this sequence was used as a single-locus probe. Two hypervariable loci were detected on Southern blots. Formal genetic investigations for the two loci were performed in order to estimate the allele frequencies. Locus 1 shows an individual-specific banding pattern with an autosomal-codominant inheritance and can be used for forensic investigations. Locus 2 also represents a polymorphic pattern, but the inheritance is not according to the Mendelian rules. Probably we have detected a highly mutagenic locus in the human genome.

TGGE and HIEF: a comparison of two methods in the detection of carriers of the Z mutation in the alpha-1-antitrypsin gene.

Alpha-1-antitrypsin (alpha-1-AT) deficiency can lead to juvenile liver cirrhosis and lung emphysema in adulthood. The deficiency Z allele is caused by a base transition. Temperature gradient gel electrophoresis (TGGE) and hybrid isoelectric focusing (HIEF) were used to detect carriers of the Z mutation of the alpha-1-AT gene. The resulting data were compared. To verify carriers at the sequence level, a manual nonradioactive sequencing strategy was established. Among our sample of carriers of the Z mutation, two were not detected by HIEF that could be identified by TGGE. DNA of all TGGE identified individuals harboring the Z mutation of the alpha-1-AT gene were sequenced nonradioactively. All carriers harbored a G to A transition at position 11.940. This mutation is described to cause the altered protein.