Successful human islet isolation utilizing recombinant collagenase

The enzymatic dissociation of acinar tissue by collagenase is a substantial step in the isolation of pancreatic islets. Although essential collagenase components have been purified, the variability in the activity of different batches limits long-term reproducibility of isolation success. The utilization of purified recombinant proteases would solve this problem. In the present study, pancreases from multiorgan donors were dissociated by means of digestion-filtration using either Liberase HI (n = 51) or a recombinant collagenase blend (n = 25). No significant differences were found regarding islet yield before and after purification, the percent of exocrine-attached islets, and final purity. However, the ratio between islet equivalents and islet numbers indicated a lesser fragmentation in islets isolated with recombinant collagenase (P < 0.01). In contrast, viability was slightly higher in islets isolated with Liberase (92.3 +/- 0.8 vs. 85.6 +/- 2.9%; P < 0.05). Insulin release during static glucose incubation was not different between experimental groups. Islet transplantation into diabetic nude mice resulted in sustained normoglycemia in either group until the graft was removed. These results demonstrated that viable human islets can be isolated using recombinant collagenase. Final optimization of this enzyme blend would offer continuous reproducibility of isolation success.     

Serial proton MR spectroscopy and diffusion imaging findings in HIV-related herpes simplex encephalitis

We report the case of pathologically proved atypical herpes simplex encephalitis (HSE) in a 40-year-old male patient with AIDS who was followed up by MR imaging, which included diffusion-weighted imaging and proton MR spectroscopy ((1)H-MRS). MR revealed sparing of hippocampi and limbic cortices, necrosis of both cingulate gyri, and cerebellar involvement. Increased diffusivity and severe metabolic alterations were compatible with biopsy findings of necrotizing inflammation. Clinical recovery corresponded with partial metabolite and diffusion normalization and a myo-inositol increase that indicated evolving gliosis formation further corroborated by immunohistochemistry results. (1)H-MRS and diffusion-weighted imaging may both support the diagnosis of HSE in patients with AIDS and help in the follow-up of necrotizing inflammation.     

Evidence-based medicine: its effect on treatment recommendations as illustrated by the changing role of postmastectomy irradiation to treat breast cancer

PURPOSE: To illustrate the effect that the quality of evidence has on clinical practice, we examined how the role of radiotherapy in treating breast cancer has changed over the years as the quality of evidence evolved from anecdotal evidence based on expert opinion to randomized clinical trials and meta-analyses. METHODS: We searched the medical literature for key randomized studies and meta-analyses that have influenced the clinical use of postmastectomy irradiation since the first randomized trials in breast cancer in the 1950s. We discuss how clinical practice changed based on the outcomes of these trials, and then discuss the quality of those trials based on the criteria currently used to assess evidence from randomized trials (CONSORT) and meta-analysis (QUORUM). RESULTS: Evidence published from the early trials and meta-analyses on the role of postmastectomy irradiation had a strong effect on clinical practice. Examination of these studies, however, continues to show significant flaws in trial design that, by today's evidence-based standards, would not meet standards of quality. CONCLUSION: The quality of evidence has a strong effect on shaping clinical practice and needs to be continually assessed. Current guidelines developed to critique both individual randomized trials and meta-analyses are helping to establish high standards for trial design and interpretation. Evidence from older trials that were not guided by well-developed guidelines need to be reviewed, particularly when results from those trials are continually updated and used to generate evidence on which to base current clinical practice.     

In vitro and in vivo antitumor properties of tetrakis((trishydroxy- methyl)phosphine)gold(I) chloride

A novel hydrophilic gold compound, tetrakis((trishydroxymethyl)phosphine)gold(I) chloride 1, has been investigated for its antitumor properties. In vitro studies demonstrate that 1 is active against HCT-15, AGS, PC-3, and LNCaP tumor cells. Cell cycle analysis of the HCT-15 cells by flow cytometry revealed elongation of the G1 phase of the cell cycle leading to growth inhibition. Administration of 1 to Balb/C mice inoculated with syngenic meth/A cells demonstrated statistically significant dose-dependent survival time.     

Syntheses of 2, 5-dialkylfuran and tetrahydrofuran carbinols and their cytotoxic activity

2, 5-Dialkylfuran and tetrahydrofuran compounds as structural elements of Annonaceae acetogenins like Asitrocinwere synthesized starting from furfural by Grignard reactions, lithiation of the furan ring and addition of aliphatic aldehydes. Hydrogenation of the furan rings over Pd-catalyst gave the corresponding tetrahydrofurans. All resulting compounds showed no or rather less antimicrobial activity against grampositive, gram-negative bacteria and fungi compared to tetracycline or clotrimazol but high cytotoxic activity against HL 60 cell line determined in the MTT assay.     

Relation of left ventricular thickness to age and gender in hypertrophic cardiomyopathy

Left ventricular (LV) wall thickening is the most consistent clinical marker of hypertrophic cardiomyopathy (HC), and characteristically increases substantially during adolescence. In this study, we used 2-dimensional echocardiography to develop a cross-sectional profile of LV wall thicknesses in adult patients with HC. We studied a regional community-based cohort of 239 consecutively enrolled patients (aged 18 to 91 years). On average, maximum LV wall thickness decreased relative to increasing age (p = 0.007) within 4 age groups: 22.8 +/- 5.1 mm (18 to 39 years) to 22.1 +/- 5.1 mm (40 to 59 years) to 21.1 +/- 3.7 mm (60 to 74 years) to 20.8 +/- 3.6 mm (>or=75 years). The LV thickness index (summation of wall thicknesses in all 4 segments) also decreased with age (p = 0.017): 63.0 +/- 12.2 mm to 59.8 +/- 11.9 mm to 58.3 +/- 10.4 mm to 57.9 +/- 9.8 mm. Decreasing magnitude of LV hypertrophy was independently associated with increasing age, but not with other relevant disease variables, such as symptoms and outflow obstruction. However, when separated by gender, this inverse relation between age and LV wall thickness was statistically significant only for women (p = 0.007). In conclusion, in an unselected HC cohort, cross-sectional analysis showed a modest but statistically significant inverse relation between age and LV hypertrophy that was largely gender-specific for women. This association constitutes another facet of the natural history of this complex and heterogenous disease and may reflect disproportionate occurrence of premature death in young patients with HC with marked hypertrophy or possibly gradual LV remodeling.     

Human prefrontal and sensory cortical activity during divided attention tasks

In our natural environment, the ability to divide attention is essential since we attend simultaneously to a number of sensory modalities, e.g., to visual and auditory stimuli. In this study, functional magnetic resonance imaging (fMRI) was used to study brain activation while a divided attention task was performed. Brain activation was also assessed under selective attention. Fourteen healthy male subjects aged between 19 and 28 years underwent fMRI studies using gradient EPI sequences. Cingulate activation was evident in all attention tasks. Focusing attention on one modality (visual or auditory) increased the activity in the corresponding primary and secondary sensory area. When attention is divided between both modalities, the activation in the sensory areas is decreased, possibly due to a limited capacity of the system for controlled processing. Left prefrontal activation, however, was evident selectively during the divided attention task. The present results suggest that this area may be important in the execution of controlled processing when attention is divided between two sources of information. These results support the view that the prefrontal cortex is involved in the central executive system and controls attention and information flow.     

Role of Fas and granule exocytosis pathways in tumor-infiltrating T lymphocyte-induced apoptosis of autologous human lung-carcinoma cells

We have isolated a cytotoxic T lymphocyte (CTL) clone, Heu161, that reacts specifically with the human autologous lung carcinoma cell line IGR-Heu. We first demonstrated that IGR-Heu lacked Fas-receptor expression and was resistant to CD95-induced apoptosis. To further elucidate the role of Fas in tumor immune surveillance, we have stably transfected IGR-Heu with a Fas-expression vector and isolated CD95-sensitive and -resistant clones. Our data indicated that the resistance of 2 selected Fas-transfected clones to CD95-mediated lysis correlated with down-regulation of caspase-8 or its lack of cleavage and subsequent activation. All Fas transfectants, either sensitive or resistant to anti-Fas agonistic antibody, were as efficiently lysed by the CTL clone as the parental cell line. In addition, neither anti-Fas-blocking antibody nor Fas-Fc molecule inhibited T-cell lysis of Fas-sensitive tumor clone. This cytotoxicity was extracellular Ca(2+)-dependent and abolished in the presence of EGTA, indicating that it was mainly granzyme-mediated. Interestingly, although the caspase inhibitor z-VAD-fmk had no effect on tumor-cell lysis, it efficiently blocked target DNA damage triggered by autologous CTLs via the granule exocytosis pathway, indicating that the latter event was caspase-dependent. The present results suggest that lung carcinoma-specific CTLs use mainly a granule exocytosis-dependent pathway to lyse autologous target cells and that these effectors are able to circumvent alteration of the Fas-triggered intracellular signalling pathway via activation of a caspase-independent cytoplasmic death mechanism.     

Lymph Edema of the Lower Extremities after Lymphadenectomy and Radiotherapy for Cervical Cancer

PURPOSE: To assess the incidence of clinical lymph edema after lymphadenectomy and postoperative radiotherapy (RT). PATIENTS AND METHODS: From 1994-2002 192 patients with risk factors for recurrence received radiotherapy with FIGO I (58.8%), II (35.4%), III (4.2%) or IV (1.6%). RT consisted of teletherapy (10.4%), brachytherapy of the vaginal vault (20.8%) or a combination of both (68.8%). Additional chemotherapy was given in 69 patients (35.9%). Surgery comprised laparoscopically assisted radical vaginal hysterectomy (LARVH) (35.4%), radical abdominal hysterectomy (RAH) (48.4%), simple hysterectomy (HE) (11.5%) or exenteration (4.7%). RESULTS: 73 patients had lymph node metastases, 119 had negative lymph nodes. In patients with LARVH 6-74 (median 30) lymph nodes were removed, with RAH 3-70 (median 21 lymph nodes), and after HE or exenteration 5-50 (median 13 lymph nodes). 90 patients had 25 or less lymph nodes removed, 83 patients more than 25 lymph nodes removed. Prognostic factors, such as age, FIGO stages, histologic grading and type of histology were well balanced in these cohorts. 45 (23.4%) of all patients developed clinically relevant lymph edema of the lower limb with a median latency of 11 (1-121) months. When 25 or less lymph nodes were removed 17.8% of patients developed leg edema, when more than 25 lymph nodes were removed 32.5% of patients were diagnosed with lymph edema (p = 0.025). Radiotherapy and chemotherapy had no influence on the incidence of leg edema. Overall survival at 5 (10) years was independent of number of lymph nodes removed. CONCLUSION: The data suggest increasing rates of leg edema with increasing number of lymph nodes dissected independent of the type of radiotherapy and chemotherapy performed. The lymph node sampling policy should be planned carefully in respect to minimize the risk of leg lymph edema.