Purification of human kallikrein 6 from biological fluids and identification of its complex with alpha(1)-antichymotrypsin

BACKGROUND: Human kallikrein 6 (hK6) is significantly increased in serum in many patients with ovarian cancer and may have a role in amyloid precursor processing and Alzheimer disease. The forms of hK6 in biological fluids are poorly characterized. METHODS: hK6 protein was immunoaffinity-purified and positively identified by Western blotting, N-terminal sequencing, and mass spectrometry. hK6 in cerebrospinal fluid (CSF), milk, ascites, and serum was size-fractionated by chromatography and then measured by a highly sensitive and specific immunoassay. Hybrid assays were performed to detect the possible interactions between hK6 and proteinase inhibitors in CSF, milk, ascites fluid, and serum. RESULTS: N-Terminal sequencing identified hK6 in the proform in both CSF and milk. hK6 exists in two forms in milk and ascites fluid: a free form with a molecular mass of approximately 25 kDa and a higher molecular mass form. Hybrid sandwich assays (capture antibody for hK6 and detection antibody for inhibitors), utilizing a panel of known serine protease inhibitors, indicated that alpha(1)-antichymotrypsin forms a complex with hK6 in milk and ascites fluid. Only the free form of hK6 was detected in CSF and serum. CONCLUSIONS: hK6 exists mainly as a proenzyme in milk and CSF. A fraction of this enzyme is partially complexed with alpha(1)-antichymotrypsin in milk and ascites fluid of ovarian cancer patients.     

Effects of naloxone on rewarding and aversive brain sites

The effects of naloxone on the rewarding and aversive properties of brain stimulation derived from the ventral tegmental area and the nucleus reticularis gigantocellularis, respectively, were assessed in rats, based on the following measures-the current threshold for latency to escape aversive nucleus reticularis gigantocellularis stimulation, the frequency threshold for rewarding ventral tegmental area stimulation, and the frequency threshold for self-stimulation obtained from delivery of concurrent ventral tegmental area and nucleus reticularis gigantocellularis stimulation, before and after three systemic doses of naloxone (0, 10, and 20mg/kg); in the latter case, the stimulation trains were interdigitated with an interpulse interval of 2 ms. Initially, thresholds for concurrent stimulation were elevated relative to the values obtained for ventral tegmental area stimulation alone, returning to baseline values only when the nucleus reticularis gigantocellularis stimulation no longer induced escape. After each pairing of the two sites, the current threshold for escape gradually increased until the maximum value administered, 700 microA, at which point aversive responses were no longer observed. This required very few pairings, between one and five trials across animals. Drug tests were then begun and produced a significant dose-response threshold increase across animals, without reinstating the latency to escape nucleus reticularis gigantocellularis stimulation. These findings are discussed in terms of a dissociation between the analgesic and rewarding properties of ventral tegmental area stimulation.     

Dall-Miles plates for periprosthetic femoral fractures. A critical review of 16 cases

Fourteen patients with 16 periprosthetic femoral fractures around hip replacement were treated with Dall-Miles plates between June 1996 and February 2000.There were 10 Vancouver B3, three B1 and three type C fractures. In addition to a Dall-Miles plate, two of the fractures (one B3 and one C type) were also stabilised with one strut graft and nine B3 fractures were revised with impaction grafting.Of the three B1 fractures treated with plates, two failed through fracture of the plate. A further two patients with B3 fractures treated with plates also failed with fracture of the plate. Failure of these plates occurred within 6 months of surgery.All non-unions and fixation failures in this series were in cases where the femoral component did not bypass the most distal fracture line by at least two cortical diameters.The Dall-Miles plates and cable system alone is insufficient for the treatment for periprosthetic femoral fractures. It must be supplemented with additional intramedullary or extramedullary fixation.     

Prevalence of age-related macular degeneration in Greece: the Thessaloniki Eye Study

PURPOSE: To estimate the age- and gender-specific prevalence of age-related macular degeneration (AMD) in a population-based sample of subjects 60 years of age or older in Thessaloniki, Greece. DESIGN: Cross-sectional population-based study. METHODS: A total of 2554 subjects randomly identified from municipality registers in Thessaloniki underwent a complete ophthalmologic examination. RESULTS: Of the 3617 eligible subjects, 2554 (71%) participants completed a clinical examination. The prevalence was 2.5% for AMD, 1.4% for neovascular (NV) AMD, and 1.3% for geographic atrophy (GA). Bilateral AMD was present in 40% of AMD cases. The prevalence of AMD, NV, GA, and bilateral AMD increased with increasing age (P< .0001). No gender-specific statistically significant differences were noted. Of all participants with AMD, 67% were unaware of the diagnosis. CONCLUSIONS: The prevalence of AMD in Greece is comparable to that in other Caucasian populations. A dramatic increase in prevalence in people older than 80 years of age was observed.     

Drug therapy for atrial fibrillation

Although the maintenance of sinus rhythm would be the ideal scenario for patients with atrial fibrillation (AF), recent randomised trials have questioned the value of this approach. A careful interpretation of their results showed the limited efficacy of currently available antiarrhythmic drugs in maintaining sinus rhythm, as well as their potentially serious side effects. Therefore, it is imperative to develop safer and more effective drugs for AF. Based on our improved understanding of the pathophysiology of AF and the mechanism of action of antiarrhythmic drugs, significant efforts are being made to develop new antiarrhythmic agents that would prevent electrophysiological remodelling, would be selective for the atria and, therefore, would not prolong ventricular repolarisation, thus lacking any proarrhythmic effect.     

The effect of different 'pass-fail' criteria on the results of a newborn hearing screening program

'Pass' criteria in newborn hearing screening programs are important, since they affect the operating characteristics of the programs. In the present study, we intended to compare the results of two screening procedures, using different 'pass' criteria, in two samples from the same pool of screened newborns. The subjects were divided into two study groups, screened consecutively during 6 months. Testing and all procedures were exactly the same in both groups, differing only in the 'pass' criteria. In the first group a signal-to-noise ratio of at least 3 dB in the frequency bands of 1-2, 2-3 and 3-4 kHz was considered necessary for a 'pass', whereas a signal-to-noise ratio > or =6 dB was used in the second group, at the same frequency bands. During the period of the study, no other minor or major modification of the protocol was applied. The comparison of the screening predischarge results between the two groups showed no statistically significant differences in the 'pass-refer' results. Thus, it appears that the 3-dB signal-to-noise ratio is as valid as the 6-dB criterion, and it may be confidently used, especially in settings where rescreening is not available.     

Prognostic implications of the immunohistochemical expression of human kallikreins 5, 6, 10 and 11 in renal cell carcinoma.

Human kallikreins 5, 6, 10 and 11 (hK5, 6, 10 and 11) are expressed by many normal tissues, and it has been suggested that they may represent candidate tumor-diagnostic or -prognostic markers. In patients with renal cell carcinoma (RCC), outcome is unpredictable despite the use of conventional prognostic factors. The aim of this study is to evaluate the immunohistochemical expression and the prognostic value of the above kallikreins in RCC. The study comprised 95 patients who underwent radical nephrectomy for RCC. The median follow-up period was 60 months (range 1-180 months). Fifty-seven RCC cases were immunostained for hK5, 70 for hK6, 70 for hK10 and 69 for hK11. The streptavidin-biotin-peroxidase method of immunostaining was performed using anti-hK5, anti-hK6, anti-hK10 and anti-hK11 monoclonal and polyclonal antibodies. The immunohistochemical expression of these kallikreins was correlated with tumor size, histological type, histological malignancy according to the Fuhrman four-grade scale, mitotic index, pathological stage and disease survival. For the statistical analysis, four grades were collapsed into two by which RCC cases were categorized as low malignant (LM) and high malignant (HM). In the normal renal parenchyma adjacent to the tumors, the renal tubular epithelium showed a cytoplasmic expression of all four kallikreins. In RCC, immunohistochemical expression was decreased: 33 of 57 cases (58%) were positive for hK5, 27 of 70 (39%) for hK6, 46 of 70 (66%) for hK10 and 32 of 69 (46%) for hK11. A statistically significant positive correlation was observed among the immunohistochemical expression of all kallikreins. HM-RCC expressed all kallikreins in a higher percentage of cases than LM-RCC, but statistically significant differences were only observed for hK6 and hK10 (55 vs. 27%, p = 0.016, and 79 vs. 56%, p = 0.044, respectively). hK6 and hK11 expression showed a positive correlation to pathological stage: hK6 with both Robson and TNM 2002 staging systems (p = 0.010 and p = 0.017, respectively), and hK11 only with the Robson staging system (p = 0.045). In both the Kaplan-Meier and the univariate Cox regression analyses, hK6 expression was negatively correlated with disease-specific survival (p = 0.05 and p = 0.038, respectively). In univariate analysis, nuclear grade, Robson stage and TNM stage also correlated with disease-specific survival. However, in the multivariate analysis, TNM stage was the only independent prognostic factor. In conclusion, although the immunohistochemical expression of hK5, hK6, hK10 and hK11 was downregulated in RCC, tumors of high grade and late stage expressed one or more of the above kallikreins in a higher percentage of cases, and hK6 may predict a poor disease outcome in RCC.