Device for filamentous fungi growth monitoring using the multimodal frequency response of cantilevers.

Filamentous fungi cause opportunistic infections in hospital patients. A fast assay to detect viable spores is of great interest. We present a device that is capable of monitoring fungi growth in real time via the dynamic operation of cantilevers in an array. The ability to detect minute frequency shifts for higher order flexural resonance modes is demonstrated using hydrogel functionalised cantilevers. The use of higher order resonance modes sees the sensor dependent mass responsivity enhanced by a factor of 13 in comparison to measurements utilizing the fundamental resonance mode only. As a proof of principle measurement, Aspergillus niger growth is monitored using the first two flexural resonance modes. The detection of single spore growth within 10 h is reported for the first time. The ability to detect and monitor the growth of single spores, within a small time frame, is advantageous in both clinical and industrial settings.

Exposure to the anti-TNF-alpha drug thalidomide induces apoptotic cell death in human T leukemic cells.

TNF-alpha is a pro-inflammatory cytokine that plays a key role in disorders due to HIV-1 infection and replication such as Kaposi sarcoma, wasting, aphthous ulcerations and progression to AIDS. The controversial drug thalidomide is anti-inflammatory, anti-angiogenic and a selective inhibitor of TNF-alpha that is being studied as a treatment for HIV-1-related disorders, immune disorders and cancer. The cellular and molecular mechanism of thalidomide is unclear despite renewed clinical interest in the drug. Previous data from this laboratory indicate that thalidomide decreases cell growth and cell-cell interactions of human T leukemic cells. The specific aim of the present study is to determine whether thalidomide administration induces cell death via apoptosis. Low dose thalidomide treatment of human T leukemic cells exhibited rapid increases in caspase-3 activity, annexin V-FITC binding and DNA disintegration that is characteristic of apoptosis. These data indicate that low doses of thalidomide signal human T leukemic cells to die by apoptosis, which is a possible method of altering inflammatory cells and inflammatory activities.

CYP17 genotype and breast cancer risk.

The MspAI polymorphism in the 5' untranslated region of CYP17 has been evaluated as a breast cancer risk factor in a hospital-based case-control study in New York City. The study population consisted of 363 women [123 breast cancer patients and 240 patient controls (123 benign breast disease without atypical hyperplasia, 117 women without breast disease)]. There were 224 Caucasians (76 cases, 148 controls), 55 African-Americans (20 cases, 35 controls) and 84 Hispanics (27 cases, 57 controls); 142 premenopausal women and 221 postmenopausal women. Consistent with a previous report (Feigelson et al., Cancer Res., 57: 1063-1065, 1997) we found no evidence to implicate the minor variant (restriction site present allele, designated A2) as a breast cancer risk factor. Furthermore, we sought evidence to implicate the minor variant of CYP17 in the development of more aggressive breast cancers (n = 38/121) as had been reported previously. Although confidence intervals (CI) overlap, the data presented here do not provide support for previously reported findings (odds ratio, 0.9; 95% CI, 0.4-2.0; n = 38 versus odds ratio, 2.5; 95% CI, 1.1-5.2; n = 40). Clearly this question needs to be resolved in a larger study. No evidence was found to support the contention that inheritance of the minor variant is a predictor of early age at menarche. Allelic frequencies between different ethnic groups were not found to be different with the exception of Hispanic controls, in which the genotypic distribution was not consistent with the Hardy-Weinberg equilibrium.

Histomorphometric and biochemical characterization of bone following acute severe burns in children.

Severe burns in adults is associated with an uncoupling of normal remodeling, low bone formation without reduced resorption. The risk of osteopenia that may occur under such circumstances is heightened by our detection in a cross-sectional study of low bone mass in severely burned children. We report here the acute histomorphometric and biochemical response of bone to severe burn injury, as well as bone mass in severely burned children. We enrolled 24 patients ages 5.8 to 17.5 years following burns of 63 +/- 16% (SD) body surface area. Serum and urine were collected weekly until iliac crest bone biopsy was obtained 26 +/- 10 days postburn. Seventeen of 18 patients, including 5 patients receiving growth hormone treatment to accelerate wound healing, failed to take up doxycycline in trabecular bone, and had no detectable osteoblasts at the osteoid seam, while eroded surface was normal and osteoblasts were documented by staining. Thus, bone formation was virtually absent. There was an eightfold elevation in urinary free cortisol excretion and high serum levels of acute phase reactants and interleukin-1 beta and -6. Biochemical markers of bone formation, osteocalcin, and type I procollagen propeptide were low, as were resorptive markers urinary pyridinoline and deoxypyridinoline. However, there was no correlation with resorptive surface. Mean age-related z-score for bone mass was -1.06 +/- 1.05, 40 days postburn. Immobilization and endogenous corticosteroid production may be the main factors responsible for acutely reduced bone formation while inflammatory cytokines may mediate resorption.

Relationship between intact 1-84 parathyroid hormone and bone histomorphometric parameters in dialysis patients without aluminum toxicity.

With the markedly reduced usage of aluminum salts in renal failure, parathyroid hormone (PTH) has become the major determinant of currently seen bone disease. Clinicians now must consider what PTH level should be sought. Too low a level may lead to the aplastic bone lesion (low turnover bone), and too high a level may cause osteitis fibrosa. Furthermore, conventional normal PTH levels may not be a suitable target because of the well-known resistance to PTH in uremic patients. In this report, we derive the PTH levels that best distinguish patients with low and high bone formation states from those with normal bone formation in a group of 175 dialysis patients without aluminum toxicity. Using bone histological parameters, we propose that ideally PTH levels should be maintained between 10 pmol/L (100 pg/mL) and 20 to 30 pmol/L (200 to 300 pg/mL) in chronic dialysis patients, levels two to four times the upper limit of values found in normal subjects.

Effect of temperature and host factors on the activities of pertussis toxin and Bordetella adenylate cyclase.

Pertussis toxin and adenylate cyclase toxin both contribute to the pathogenesis of whooping cough. Production of these proteins is controlled by the bvg locus, which is inactive at 25 degrees C, but at 37 degrees C produces a Vir+ phenotype. In view of the temperature dependence of virulence factor synthesis, the effects of temperature and host factors on their action were examined. The NAD glycohydrolase activity of the S1 subunit of pertussis toxin was enhanced by CHAPS, a zwitterionic detergent, with a temperature optimum of approximately 35 degrees C. Similar temperature optima for the ADP-ribosylation by pertussis toxin of transducin and recombinant Go alpha were observed. Since the temperature--activity relationship of S1 differed from that of S1 in activated holotoxin, and since S1 in activated holotoxin was more stable at 42 degrees C than was S1, it appears that S1 associated with the B oligomer components may, in fact, be an active species. Bordetella pertussis adenylate cyclase is activated by a host factor, calmodulin. In the absence of calmodulin, the temperature optimum for enzymatic activity was approximately 25 degrees C, whereas in its presence it was approximately 35 degrees C. Thus, the temperature optima for pertussis and adenylate cyclase toxins, virulence factors whose production is increased through the bvg locus at physiological temperatures, are either at or near these temperatures when stimulated by host factors.

Characterization of adenylate cyclase toxin from a mutant of Bordetella pertussis defective in the activator gene, cyaC.

Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin which is able to bind to target cells but unable to elicit cAMP accumulation. In that AC toxin from BPDE386 is able to function normally when injected artificially to an intracellular site, we conclude that the disruption of cyaC produces a defect in insertion and transmembrane delivery of the catalytic domain.

The spectrum of bone disease in end-stage renal failure--an evolving disorder.

We have assessed the bone histology in 259 chronic dialysis patients, all of whom were in the same dialysis program. All patients had bone biopsies with quantitative histomorphometry, intact parathyroid hormone (PTH) measurements, basal and deferoxamine stimulated serum aluminum levels. Results demonstrate the increased incidence of the recently described aplastic bone lesion, particularly in patients treated with peritoneal dialysis (PD). Aluminum-related bone disease is much less common than previously described, perhaps in relation to the declining use of aluminum as a phosphate binder. A different pattern of bone lesions is seen in PD as compared with hemodialysis (HD), with low turnover disorders comprising 66% of the lesions seen in PD and high turnover lesions accounting for 62% of the bone histologic findings in HD. The difference in these patterns may relate to alterations in PTH levels, as mean PTH levels in HD patients were 2-1/2 times the levels found in PD patients (P < 0.0005), while older age, higher prevalence of diabetes and a shorter duration of dialysis may also have contributed to the findings in the PD patients. We suggest that PD, perhaps by maintaining calcium at higher levels, may more effectively suppress the parathyroid gland.

Effects of 1-week head-down tilt bed rest on bone formation and the calcium endocrine system.

To understand the potential early responses of human bone and the calcium endocrine system to spaceflight, we studied 8 healthy men, aged 35-44 years before, during, and after bed rest in a -6 degrees head-down tilt model for microgravity. Based on a novel single-dose labeling schedule, average rates of bone formation in the iliac crest were reduced in 6, unchanged in 1, and increased in 1 following the bed rest period. The decrease was greatest for subjects whose daily walking miles were highest (r = -0.762, p less than 0.05, n = 7). Before a measurable increase in ionized serum calcium the sixth bed rest day, there was increased excretion of urinary calcium and sodium, evident the first 2 bed-rest days and parallel for the entire week (r = 0.92, p less than 0.001). Reduced excretion of phosphorus and 3', 5' cyclic adenosine monophosphate on the first and second bed rest days was followed by an increase in serum phosphorus by the sixth bed rest day. Depressed serum concentrations of parathyroid hormone and 1,25-dihydroxyvitamin D were manifest by the sixth and seventh bed rest days. The similarity of the response of bone and the calcium endocrine system of healthy men after only 7 days to results of longer term bed rest studies emphasizes the responsiveness of the adult human skeleton to biomechanical stimuli induced by changes in activity and/or position.

Beta 2-microglobulin deposition in bone in chronic renal failure.

Recently, there have been reports of beta 2-microglobulin (beta 2 m) related amyloid deposition in perineural and periarticular tissues in patients receiving long-term hemodialysis, but it has been rarely described in bone. We, therefore, examined previously obtained bone biopsy specimens in patients receiving long-term hemodialysis to determine the prevalence of beta 2 m deposition in bone and to assess the relationship between beta 2 m deposits and bone histomorphometry. We found beta 2 m deposits in bone in 8% of 224 patients examined. Bone deposition of beta 2 m was absent in patients who were on dialysis for less than six years, but was present in 19% who dialyzed longer than 10 years. beta 2 m deposits were found in specimens from the iliac crest, femoral bone, tibia, vertebra and rib. In the iliac crest beta 2 m deposition was localized predominantly to the periosteum. Among these patients with beta 2 m in iliac crest periosteum, 62% had suffered a femoral neck fracture compared to only 4% of matched patients who had negative staining for beta 2 m in the iliac crest (P less than 0.001). Histologically, osteitis fibrosa seemed more common in patients positive for beta 2m than in patients negative for beta 2m deposition. We conclude that beta 2m deposition in bone is common in uremic patients who have received hemodialysis longer than 10 years. The high prevalence of femoral neck fracture in patients with beta 2m localized to the periosteum of the iliac crest suggests that this involvement may be useful to predict susceptibility to femoral fracture.

Single-dose tetracycline labeling for bone histomorphometry.

A single oral dose of tetracycline deposits in bone and is readily identified with fluorescence microscopy. Two such time-spaced labels can be used to determine bone dynamic features. This is as accurate as conventional three-day labeling periods. The simplicity, improved compliance, and substantial reduction in time it takes to prepare a patient for bone biopsy all appear to be advantageous when compared with current recommendations.

Bone aluminum accumulation in hemodialysis patients: a longitudinal perspective.

Serial bone biopsies obtained from 19 chronic hemodialysis patients asymptomatic for bone disease were examined retrospectively. We found only modest amounts of stainable bone aluminum after the first few years of hemodialysis therapy. Over the ensuing 5 to 15 years, there was a progressive increase in stainable bone aluminum (% of total bone surface): 0 to 4.9 years, 19.9 +/- 3.8% (mean +/- SEM); 5.0 to 9.9 years, 28.4 +/- 5.3%; and greater than or equal to 10.0 years, 58.0 +/- 7.7%. At final biopsy, the extent of bone surface aluminum was significantly correlated with duration of hemodialysis therapy (r = 0.54) and with bone formation rate (BFR) (r = -0.54). Patients who developed aluminum-associated bone disease did not differ from other patients in duration of hemodialysis, intake of 1-hydroxylated vitamin D3 compounds, or the findings on early bone biopsy. High-turnover renal osteodystrophy remains the dominant bone lesion throughout the course of hemodialysis in patients with intact parathyroid glands. In individual patients, bone histology frequently changes over time and in some patients, aluminum-associated bone disease may improve spontaneously. Bone biopsy in an asymptomatic patient who has received hemodialysis therapy for less than 5 years does not appear to be useful in predicting the subsequent appearance of aluminum-associated bone disease.

Reactivation of inhibited bone acid phosphatase and its significance in bone histomorphometry.

Despite biochemical demonstration of acid phosphatase (AcP) activation or reactivation in bone, few attempts have been made to show similar effects histochemically. Bones from growing rats, when fixed in 4% buffered formaldehyde at room temperature and demineralized in 5% formic acid, exhibited expected inactivation of AcP. The inhibited AcP, however, was reactivated by pre-incubation of sections for 1 hr at 37 degrees C in the following buffers: 0.2 M Tris, 0.2 M glycine, 0.2 M NaHCO3, or 0.1 M borax, as well as in alkaline water, but not in 0.2 M Na2HPO4 (all at pH 9). The reactivation was (a) site-specific (e.g., osteoclasts, osteoblasts, osteocytes, and cement lines), (b) temperature- and pH-dependent, (c) unaffected by OH- or SH--binding agents or by an alkaline phosphatase inhibitor, and (d) inhibited completely by 10 mM Na2HPO4. The reactivation process, much simplified and/or more effective than with the methods previously reported, was observed in all 83 human biopsy bones embedded in methyl methacrylate and in human bones stored in cold buffered formaldehyde for 7 months. This study demonstrates a unique method for reactivating and thus localizing the inhibited AcP in bones, and suggests possible applications in bone histomorphometry.

Osteomalacia and aplastic bone disease in aluminum-related osteodystrophy.

The bone histology in patients with chronic renal failure and aluminum-related bone disease does not always show the excess accumulation of unmineralized osteoid (matrix) characteristic of osteomalacia. Frequently, bone aluminum accumulation is associated with normal or reduced amounts of unmineralized osteoid and low bone formation and is referred to as aplastic bone disease. In this study, we compared static and dynamic bone histomorphometric parameters and plasma PTH and aluminum levels in 12 patients with osteomalacia and 18 patients with aplastic bone disease who had been receiving dialysis for the same duration to determine if the difference in osteoid accumulation in these 2 lesions might be explained by differences in aluminum accumulation or PTH levels. The stainable bone surface aluminum level was significantly higher in the patients with osteomalacia compared to that in the group with aplastic bone [61 +/- 5% (+/- SEM) vs. 43 +/- 4%; P less than 0.02]. The rates of bone apposition and bone formation were lower in the group with osteomalacia (P less than 0.01). Plasma amino-terminal PTH was not significantly different in the 2 groups. The increment in plasma aluminum levels after a single infusion of deferoxamine was higher in the osteomalacic group than in the aplastic group, suggesting that the patients with osteomalacia accumulated more total body chelatable aluminum than did those with aplastic bone disease during a comparable length of time on dialysis. We conclude that the excess unmineralized osteoid in aluminum-related osteomalacia results from the high rate of total body aluminum accumulation, which directly causes uncoupling of matrix mineralization and matrix production, independent of PTH levels. Patients with aplastic bone disease who have accumulated lesser amounts of total body aluminum fail to develop excess unmineralized osteoid because production and mineralization of matrix are more closely coupled than in the osteomalacic lesion, despite a decline in osteoblast numbers.

Early deposition of aluminum in bone in diabetic patients on hemodialysis.

Aluminum-associated bone disease is a special problem in uremic patients on hemodialysis. We have observed this disorder in uremic patients with insulin-dependent diabetes soon after the start of dialysis treatments. We therefore studied bone biopsy specimens from 18 diabetic patients on hemodialysis to determine whether aluminum accumulates on bone surfaces at an accelerated rate in diabetes. We also measured the rates of bone formation, because lower rates may enhance the accumulation of aluminum on bone surfaces. As compared with 18 nondiabetic controls with uremia who were matched for age and duration of dialysis, the patients with diabetes had a higher rate of aluminum accumulation on bone surfaces (2.1 +/- 0.7 vs. 0.4 +/- 0.2 percent per month, P less than 0.01) and a lower rate of bone formation (117 +/- 50 vs. 396 +/- 81 microns 2 per square millimeter per day, P less than 0.01). Also, the patients with diabetes whose cumulative aluminum intake exceeded 0.5 kg had higher serum aluminum levels after an infusion of deferoxamine, as compared with controls matched for aluminum intake (P less than 0.01). These measurements reflected a higher aluminum content in the whole body in patients with diabetes. We suggest that the enhanced rate of aluminum accumulation on bone surfaces in uremic patients with diabetes occurs as a result of a low rate of bone formation and an increased accumulation of aluminum in the whole body.

Comparison of parathyroid hormone assays with bone histomorphometry in renal osteodystrophy.

Bone biopsies were studied in 67 dialysis patients to determine if a PTH RIA specific for intact plasma PTH is a better predictor of osteitis fibrosa than a RIA that measures inactive carboxy-terminal/midregion plasma PTH fragments. An amino-terminal-specific antiserum that cross-reacts with intact PTH, but not midregion or carboxy-terminal fragments, and an antiserum that cross-reacts with the 44-68 region of the PTH molecule and measures both intact and midregion/carboxy-terminal PTH fragments were used in the comparisons. Plasma PTH concentrations measured by both assays correlated positively with bone formation rate, bone apposition rate, osteoblastic osteoid, osteoclast number, and marrow fibrosis. The optimum predictive value of the amino-terminal PTH assay for osteitis fibrosa was 88%, compared to 74% for the midregion PTH assay. This difference in predictive value could be attributed to the highly significant correlation of marrow fibrosis with plasma amino-terminal PTH. In conclusion, these data suggest that a PTH RIA that uses an amino-terminal-specific antiserum may be a better predictor of osteitis fibrosa in patients undergoing maintenance hemodialysis.

Aluminum-associated bone disease in chronic renal failure: high prevalence in a long-term dialysis population.

Twenty-seven asymptomatic patients treated with hemodialysis longer than 8 years (mean 12.9 +/- 3.1 years) underwent bone biopsy to determine the prevalence of aluminum-associated bone disease. None had excess aluminum exposure from the dialysate. Ten patients (37%) had aluminum-associated bone disease as defined by a bone formation rate (BFR) below normal in the presence of stainable bone aluminum that covered more than 25% of the trabecular surface. The predominant type of bone histology in this group was the aplastic lesion characterized by low bone turnover, a decreased number of osteoblasts, and lack of excess unmineralized osteoid. Osteoblastic osteoid was highly correlated with stainable surface bone aluminum (r = -.82, p less than .001). Among the dynamic bone parameters, the double-tetracycline labeled surface was a more sensitive indicator of impaired bone function than was the bone apposition rate (BAR), since half of the patients with aluminum-associated bone disease had a normal BAR. In all of the biopsies the extent of double-labeled surfaces was inversely proportional to the amount of stainable aluminum on the bone surface (r = -.71, p less than .001), whereas stainable bone aluminum did not correlate with BAR. In seven of the patients with aluminum-associated bone disease, amino-terminal PTH levels were in the normal range while only one patient had a normal plasma mid-region PTH. PTH correlated directly with osteoblastic osteoid, BFR, and double-labeled surfaces. These results indicate that long-term oral aluminum intake in hemodialysis patients results in a high prevalence of aluminum-associated bone disease.(ABSTRACT TRUNCATED AT 250 WORDS)