Metformin shows anti-neoplastic properties by inhibition of oxidative phosphorylation and glycolysis in epidermolysis bullosa-associated aggressive cutaneous squamous cell carcinoma.

BACKGROUND: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target. OBJECTIVES: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC. METHODS: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo. RESULTS: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival. CONCLUSIONS: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

B-cell activating factor BAFF reflects patients' immunological risk profile after kidney transplantation.

The B-cell activating factor BAFF plays an important role in the development and maturation of B-lymphocytes, which can contribute to the generation of donor-specific antibodies and thus may influence graft function and graft survival. Inconsistent data on the role of BAFF levels after renal transplantation for the formation of donor-specific antibodies and the contribution for allograft rejection exist. The aim of the current study was to determine to what extent the degree of pre-immunization is reflected by each patient's BAFF levels before transplantation and in the follow-up. Furthermore, the impact of BAFF on allograft rejection frequency as well as severity and resulting allograft function over time was analyzed. Additionally, the impact of viral infections on BAFF levels after transplantation - as a potential confounder - was examined. For this purpose, a group of pre-sensitized patients (PRA>0%, (52±24% on average), n=40) was compared with non-sensitized patients (PRA=0%, n=62) and in a subsequent analysis stratification in accordance to the detected BAFF level was performed. Pre-sensitized patients had significantly higher BAFF levels before transplantation and suffered significantly more often from early steroid-resistant, mainly antibody-mediated rejections. A result which was confirmed also in highly sensitized patients with PRA levels >50%. Additionally, in the follow-up patients with either rising BAFF levels over time or BAFF levels above the median also had significantly more often antibody mediated rejections. Additionally, patients with BAFF levels above detected median even displayed impaired creatinine values as well as an induced eGFR slope up to month 48 after transplantation. The occurrence of viral infections (CMV, BKV) was only an additional influencing factor in the absence of concomitant allograft rejections. Therefore, the B-cell proliferation factor BAFF appears not only to reflect the immunological risk profile of patients in the context of kidney transplantation, it may possibly be further developed as a predictor of patients with an increased risk profile for subsequent allograft rejection and impaired allograft function.

Actinomycin D induces p53-independent cell death and prolongs survival in high-risk chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most prevalent lymphoid malignancy in the elderly of the Western world. Although treatment options have improved over the past two decades, 10-15% of patients still have a poor prognosis and are often resistant to therapy. Aberrations in the p53 pathway, such as a deleted (del17p13) or mutated p53 gene, are highly enriched in this class of patients. In an extensive screen for p53-independent apoptosis inducers, actinomycin D was identified from 1496 substances and shown to induce apoptosis in primary CLL cells derived from high-risk patients including those with aberrant p53, revealing a novel p53-independent mechanism of action. Both pro-survival genes BCL2 and MCL1 are targeted by actinomycin D, in contrast to fludarabine the backbone of current treatment schedules. In the well-established TCL1 transgenic mouse model for high-risk CLL, actinomycin D treatment was more effective in reducing tumor load than fludarabine, with no evidence of resistance after three treatment cycles and an overall survival increase of over 300%. Tumor load reduction was coupled to BCL2 downregulation. Our results identify the clinically approved compound actinomycin D as a potentially valuable treatment option for CLL high-risk patients.

Development of CLL in the TCL1 transgenic mouse model is associated with severe skewing of the T-cell compartment homologous to human CLL.

Chronic lymphocytic leukemia (CLL) cells require complex microenvironmental and immunologic interactions to survive and proliferate. Such interactions might be best recreated in animal models; however, this needs extensive verification. We therefore investigated the composition of the T-cell compartment in the Eµ-TCL1 transgenic mouse, currently the most widely used murine model for CLL. Immunophenotyping and transplant approaches were used to define T-cell subsets at various stages of CLL. Analogous to human CLL, we observed a skewing of T-cell subsets from naive to antigen-experienced memory T cells that was more pronounced in lymph nodes than in blood. Transplantation of CLL into non-transgenic recipients was feasible without immunosuppression in a pure C57BL/6 background and resulted in the prominent skewing of the T cells of the recipient mice. Both in spontaneously developed CLL and in the transplantation setting, a loss in T-cell receptor diversity was observed, with a relevant number of clonal T-cell populations arising. This suggests that antigen-dependent differentiation toward the T memory pool is initiated by murine CLL cells. In summary, we validate the TCL1 transgenic mouse model for analysis of T-cell phenotypes and suggest a CLL-dependent antigen-driven skewing of T cells in these mice.

[Smelling diseases? A short review on electronic noses]. / Krankheiten erriechen? Eine kurze Übersicht über elektronische Nasen.

Non-invasive pulmonary diagnostics is a promising and interesting field in respiratory medicine. Beside exhaled breath condensate, there is an increasing interest in alternative and faster techniques such as electronic noses (EN). EN aim to mimic or improve the sense of smelling. Different types of EN have been employed in research so far. In addition to ion mobility spectrometry and mass spectrometry, ENs that consist of various biopolymer sensors for the sensing of volatile organic compounds (VOCs) have been tested. VOCs bind to the sensors depending on size, structure, hydrogen binding and polarity. This leads to physical alterations, e. g., swelling resulting in a change of resistance. The smell print represents composite patterns in contrast to single compounds, and the distinction between different categories is achieved by pattern recognition algorithms. Other types of EN like mass spectrometry and ion mobility spectrometry are capable of identifying even single analyte fractions provided that their characteristics have been saved in data repositories. The non-invasive nature, onsite availability and relatively cheap sampling are advantages of ENs that underly the increasing interest in their use for medical purposes. Some promising results have already been published. This review aims to describe the state of the art in brief form.

[Comparison of four identical electronic noses and three measurement set-ups]. / Vergleich von vier baugleichen elektronischen Nasen und drei Messaufbauten.

BACKGROUND: Volatile organic compounds (VOCs) can be used as biomarkers in exhaled air. VOC profiles can be detected by an array of nanosensors of an electronic nose. These profiles can be analysed using bioinformatics. It is, however, not known whether different devices of the same model measure identically and to which extent different set-ups and the humidity of the inhaled air influence the VOC profile. METHODS: Three different measuring set-ups were designed and three healthy control subjects were measured with each of them, using four devices of the same model (Cyranose 320™, Smiths Detection). The exhaled air was collected in a plastic bag. Either ambient air was used as reference (set-up Leipzig), or the reference air was humidified (100% relative humidity) (set-up Marburg and set-up Munich). In the set-up Marburg the subjects inhaled standardised medical air (Aer medicinalis Linde, AGA AB) out of a compressed air bottle through a demand valve; this air (after humidification) was also used as reference. In the set-up Leipzig the subjects inhaled VOC-filtered ambient air, in the set-up Munich unfiltered room air. The data were evaluated using either the real-time data or the changes in resistance as calculated by the device. RESULTS: The results were clearly dependent on the set-up. Apparently, humidification of the reference air could reduce the variance between devices, but this result was also dependent on the evaluation method used. When comparing the three subjects, the set-ups Munich and Marburg mapped these in a similar way, whereas not only the signals but also the variance of the set-up Leipzig were larger. CONCLUSION: Measuring VOCs with an electronic nose has not yet been standardised and the set-up significantly affects the results. As other researchers use further methods, it is currently not possible to draw generally accepted conclusions. More systematic tests are required to find the most sensitive and reliable but still feasible set-up so that comparability is improved.

Stone treatment and coagulopathy.

OBJECTIVE: The aim of this retrospective study was to evaluate treatment protocols and results of upper tract stone treatment in patients with clotting disorders. METHODS: In a 6-year period, 6,827 stone interventions (ESWL or endourologic procedures) were performed in 5,739 patients. Thirty-five (0.61%) patients suffered from a variety of systemic clotting disorders or were anti-coagulated. Clotting disorders were corrected by specific therapy prior to any intervention. A total of 76 interventions were performed consisting of ESWL, ureteroscopy (URS), percutaneous nephrolithotomy (PNL), ureteric stenting or percutaneous nephrostomy. RESULTS: All patients became stone-free within 3 months or had clinically insignificant residual fragments. Severe complications were observed in 10/76 (13.1%) interventions. ESWL was successful in 88.9% (16/18) of patients, but associated with a 33.3% (6/18) complication rate; 27.8% (5/18) of patients required auxiliary procedures. URS and PNL were successful in all cases and complications occurred in 0% (0/7) and 33% (1/3) of patients, respectively. Time to complete stone clearance after ESWL was 32.0+/-49.3 days compared with a mean of 19.4+/-28.6 days in a non-coagulopathy control group; no difference was observed for endourologic procedures. Average costs of treatment in patients undergoing ureteroscopy was higher in patients with coagulopathy (4,611 versus 2,342); however, the difference was less pronounced compared with ESWL (6,070 versus 1,731). CONCLUSION: Patients with coagulopathy have a higher rate of complications despite apparently normal clotting parameters during treatment and hospitalisation was prolonged. The efficacy of ESWL was lower in patients with coagulopathy and we currently favour endoscopic procedures for stone removal in this patient group.

Evolutionary stability concepts for N-species frequency-dependent interactions.

The classical static concept of an evolutionarily stable strategy (ESS) for a single species gives rise to two new notions when there are more than two species (called an N-species ESS and RL-stability). The paper relates these to the dynamic stability of monomorphic and polymorphic evolutionary systems. It is shown that RL-stability implies the global asymptotic stability of either system with or without mutations. However, the N-species ESS only implies stability of the monomorphic system.

The impact of urinary extravasation after radical retropubic prostatectomy on urinary incontinence and anastomotic strictures.

OBJECTIVE: The aim of this study was to determine the relevance of urinary extravasation as proven by cystogram 18 days after radical retropubic prostatectomy for the degree of postoperative urinary incontinence and the incidence of anastomotic strictures. PATIENTS AND METHODS: A total of 225 patients underwent radical retropubic prostatectomy at our institution during a 30-month period, 215 of whom received a cystogram a mean 18 days following surgery. Three and 6 months after surgery these 215 patients were evaluated regarding the degree of urinary incontinence and the presence of anastomotic strictures. RESULTS: The cystogram demonstrated a watertight anastomosis in 89% (n = 195; group I), the remaining 11% (n = 24; group II) showed urine extravasation. Groups I and II were comparable with respect to age, preoperative serum levels of prostate-specific antigen (PSA), tumor grade and pathological staging. Six months after surgery, there was no statistically significant (p > 0.05) difference between both groups regarding the degree of urinary incontinence and the presence of anastomotic strictures. CONCLUSIONS: The presence of urine extravasation 18 days after radical retropubic prostatectomy has no impact on postoperative urinary incontinence and the incidence of anastomotic strictures. Based on these data it is not indicated to leave the catheter in situ beyond that point of time.

Convergence of multilocus systems under weak epistasis or weak selection.

The convergence of multilocus systems under viability selection with constant fitnesses is investigated. Generations are discrete and nonoverlapping; the monoecious population mates at random. The number of multiallelic loci, the linkage map, dominance, and epistasis are arbitrary. It is proved that if epistasis or selection is sufficiently weak (and satisfies a certain nondegeneracy assumption whose genericity we establish), then there is always convergence to some equilibrium point. In particular, cycling cannot occur. The behavior of the mean fitness and some other aspects of the dynamics are also analyzed.

Frequency of urolithiasis in individuals seropositive for human immunodeficiency virus treated with indinavir is higher than previously assumed.

PURPOSE: Indinavir was approved by the Food and Drug Administration in 1996 as a human immunodeficiency type 1 protease inhibitor to treat human immunodeficiency virus infection. Prompted by the high number of patients receiving indinavir who present with renal colic at our institution, we performed a detailed investigation of the true frequency of urolithiasis during indinavir treatment. MATERIALS AND METHODS: We evaluated 105 patients with a mean age of 38.1 years who were treated with indinavir from 1996 to 1997. Before indinavir treatment was initiated all patients underwent renal ultrasonography, urinalysis, and determination of serum sodium, potassium, calcium, uric acid and creatinine. It was recommended that all patients drink 2 l of fluids daily, and all remained under continuous surveillance. RESULTS: Metabolic evaluation and ultrasonography showed no abnormality in any case. A stone episode occurred in 13 men (12.4%) as renal colic during observation. Colic recurred in 1 patient after 2 and 5 months, and in 1 after 2 months. Median duration of indinavir treatment until an acute stone episode was 21.5 weeks (range 6 to 50). A total of 12 stones passed spontaneously. Three patients underwent ureteroscopic calculous removal and 1 was treated with extracorporeal shock wave lithotripsy. CONCLUSIONS: Despite adequate patient information and compliance the rate of nephrolithiasis during indinavir therapy was 12.4%.

Multifocality of transitional cell carcinoma results from genetic instability of entire transitional epithelium.

OBJECTIVES: Multifocality of transitional cell carcinoma (TCC) has been attributed to seeding of exfoliated tumor cells or to a general sensitivity of the entire urothelium to carcinogenic stimuli. By contrast, TCC has been shown to evolve as a consequence of genetic defects and chromosomal instability. We analyzed chromosomal patterns, total DNA content, and p53 and Ki67 expression in malignant and normal transitional cells to evaluate their relationship to the development of multifocal TCC. METHODS: Included in the study were 47 patients, 16 women and 31 men, with a mean age of 70.04 years (range 37 to 83). Of 47 patients, 45 had TCC of the urinary bladder and 7 of those had synchronous ureteral involvement. Two patients had ureteral TCC and a history of TCC of the bladder. Using fluorescence in situ hybridization, numerical aberrations of chromosomes 7, 9, and 17 were detected in imprint specimens of histologically verified tumor and "normal" urothelium and were compared with static ploidy and p53 and Ki67 expression. RESULTS: Chromosome 7 was altered in 93.6%, chromosome 9 in 63.8% (including monosomy), and chromosome 17 in 87.2% of the 47 analyzed tumor and normal imprints. Differences between tumor and normal epithelium were observed in aberrational frequencies (number of cells showing chromosomal aberrations calculated on 200 cells counted, given in percentages). DNA content was aneuploid in all tumor specimens, but diploid in 20 (42.5%) of 47 normal specimens, according to lower aberration frequencies in these patients. p53 detection was positive in 82.9% of the tumor specimens and 76.6% of the normal specimens. Ki67 was positive in 87.2% of the tumor imprints and in 72.3% of the normal specimens. CONCLUSIONS: These data suggest a general genetic instability as a reason for multifocality in the entire transitional epithelium.

N-acetylneuraminic acids (nana): a potential key in renal calculogenesis.

N-Acetylneuraminic acids (NANA) promote binding of calcium ions to macromolecules and cells, increase the intrinsic viscosity of glycoproteins and facilitate gel formation in water. Since these properties are crucial in urinary calculogenesis, we evaluated NANA levels in urine and serum as well as their expression in kidney tissues. Using a modified thiobarbituric acid assay, the evaluation of free and bound NANA in 24-h urine samples revealed a ratio of 1.87 in 33 non-stone-formers but a reversed ratio of 0.84 in 41 recurrent calcium oxalate stone-formers. Time kinetics revealed a gradual rise in NANA expression until 48 h of culture and a significantly higher release into supernatants of papillary renal epithelial cells (REC) when compared with cortical REC. To examine NANA distribution in kidney tissues, paraffin-embedded biopsies from five normal and six stone-forming kidneys were labeled with the biotinylated NANA-specific lectins Maackia amurensis (MAA) and Sambucus nigra (SNA). Immunohistochemistry revealed intense luminal MAA reactivity of distal tubular REC and collecting ducts in 96.7% and 91.5% of normal and stone-forming kidneys respectively. By contrast, there was a marked difference between normal and stone-forming kidneys for SNA reactivity (17.7% vs 95%) at the same locations. Finally, the glycocalyx of recurrent stone-formers showed altered sialylglycoside linkages [alpha(2,6) instead of alpha(2,3)] that may indicate an altered REC function. Given the calcium-binding potential of NANA, their increased local concentration within the glycocalyx layer in the distal nephron may either initiate stone formation or facilitate attachment of microcrystals to REC.

Does topical instillation therapy influence chromosomal aberrations in superficial bladder cancer?

PURPOSE: Patients with a high risk for superficial bladder cancer are treated by topical immuno-or chemotherapy after transurethral resection to reduce the chance of recurrence and/or progression. The aim of this study was to analyse if cytogenetical abnormalities, which are known to be constantly related to bladder cancer, are modified or eliminated by topical immuno- or chemotherapy. MATERIALS AND METHODS: Using fluorescence in situ hybridization (FISH), the influence of topical instillation therapy with Bacillus Calmette-Gúerin (BCG) and Mitomycin C (MMC) on numerical aberrations of chromosomes 7, 9 and 17 was investigated in 25 patients with transitional cell cancer (TCC) of the bladder. Data were compared with histological and clinical outcome. Fifteen TCC patients with similar histological criteria without instillation therapy served as controls. Median follow-up was 30 +/- 2 months. RESULTS: After BCG treatment 10 of 15 patients (66.6%) developed recurrent and 2/15 (13.3%) progressive disease. Three of 15 patients (20.0%) had no evidence of disease. Numerical aberrations did not change in 8 of the 15 BCG patients (53.3%) and changed to a more aggressive pattern in 40.0% (6/15). Five of 10 MMC treated patients (50.0%) developed a recurrent tumor, 2/10 (20.0%) progressed and 3/10 (30.0%) had no evidence of disease. Four of 10 (40.0%) of these patients showed stable and 5/10 (50.0%) progressive chromosomal patterns. Only one patient in each group with primary chromosomal alterations changed to a regular diploid chromosomal pattern after therapy according to a complete clinical remission. CONCLUSION: Even though topical immuno- and chemotherapy may be useful to delay recurrence and progression, chromosomal patterns remain basically unstable.

Late urological complications and malignancies after curative radiotherapy for gynecological carcinomas: a retrospective analysis of 10,709 patients.

PURPOSE: A retrospective study was done to analyze late urological complications following curative radiotherapy of primary gynecological carcinomas. All patients were treated at a single center and with the same radiotherapeutic regimen. The incidence of other carcinomas in these patients was also evaluated. MATERIALS AND METHODS: A total of 10,709 patients was treated using combined telebrachytherapy (dosage 67.5 Gy.) during an observation period of 22 years. RESULTS: Severe late complications were seen in 1.24% (133 of 10,709) of the patients, including irradiated bladder (65 cases, mean interval since treatment 6.7 years). Complications required surgery in 118 of 133 patients with a perioperative mortality of 4.2% (5 of 118). Overall in 4.27% (457 of 10,709) of the patients another malignancy developed after (29.1%), during (26.3%) and before (44.6%) radiotherapy. Subsequent malignancies after treatment were predominantly seen in the genital region (88.4%) but they were also in the irradiated nongenital area (0.13%, 14 of 10,709). Of the latter patients 6 had urothelial bladder cancer, which represents a relative risk of 4.66 (based on the Austrian female population) to develop bladder cancer after radiotherapy for gynecological cancer. CONCLUSIONS: Late urological complications after radiotherapy of the pelvis are rare but severe. Surgical therapy of irradiated tissues has a higher complication rate compared to surgery on nonirradiated tissue.