Measurement of β2 microglobulin in dried urine spotted on filter paper by means of a forward sandwich enzyme-linked immunosorbent assay

We devised a highly sensitive forward sandwich enzyme-linked immunosorbent assay (ELISA) for estimation of the ₂-microglobulin concentration in dried urine spotted on filter paper. This method is suitable for mass screening because of the good reproducibility, satisfactory stability, low operation cost and easy sample collection. However, preliminary studies using our new method for detecting renal diseases in the Tokyo infant population did not produce good results. The high cut-off value may have been the main reason. To make this ELISA more suitable for mass screening, we plan to reduce the cut-off value, test older children and improve the extraction step.     

Effect of keratinocyte growth factor—2 on proliferation of human adult keratinocytes

OBJECTIVE: To investigate the proliferative effect of keratinocyte growth factor (KGF-2) on human adult keratinocytes. METHODS: The standard medium was keratinocyte growth medium without bovine pituitary extract (BPE), hydrocortisone or epidermal growth factor (EGF). Keratinocytes from a 48-year-old subject were cultured and seeded on dishes with standard medium of EGF in cell density of 2 x 10(4)/32 mm(2). After 24 hours, the medium was replaced by the standard medium with 0, 4, 16, 125 and 500 ng/ml KGF-2, respectively. The standard medium with EGF was used as the positive control and the standard medium without EGF or KGF-2 was used as the negative controls. The growth of keratinocytes was monitored by 3-(4,5-dimethythiazol-2-yl)-2,5 dipheyl tetrazolium bromide (MTT) assay and by photographs on days 3, 5 and 7, respectively. RESULTS: KGF-2 in concentrations of 4-500 ng/ml showed a significant proliferative effect on days 5 and 7 as compared with that of the negative controls (P < 0.01). On day 3 the cells were proliferated to 1.5-2.5-fold, on day 5 to 3-5-fold and on day 7 to 3-12-fold in KGF-2 medium as that of the negative controls. The optimal response occurred when the concentration of KGF-2 was 125 ng/ml on day 7. Cell proliferation was also consistently higher in all KGF-2 concentrations as compared with that of the positive controls. CONCLUSIONS: KGF-2 has significant effects on the proliferation of adult keratinocytes, which are more effective than that of EGF. This study supports KGF-2 can improve the healing of chronic wounds in adults in clinic.     

Night-time polyuria and urine hypo-osmolality in enuretics identified by nocturnal sequential urine sampling--do they represent a subset of relative ADH-deficient subjects?

Early morning urine osmolality was tested in two urinary specimens, one taken immediately upon awakening and the other approximately 30 min thereafter, in 52 enuretic and 15 non-enuretic children. In a follow-up study, using the same study population, urine osmolality and volume were measured sequentially at 3-h intervals at 19.00, 22.00, 01.00, 04.00 and 07.00 h. Thereafter, all enuretics were treated by intranasal DDAVP for a 6-month period. There were no differences in urinary osmolality between enuretic and non-enuretic children when comparing the two early morning specimens. Nor were there any differences between groups in urine osmolalities at 19.00, 01.00 and 07.00 h. In contrast, at 04.00 h, urine osmolality was significantly lower in 17 of 52 enuretics [designated as ADH-negative (ADH-)] compared to the remaining enuretics [designated as ADH-positive (ADH+)] and non-enuretic children (610 +/- 251 vs 995 +/- 195 and 1089 +/- 195 mosmol/kg H2O, respectively, p < 0.05). This decreased osmolality was paralleled by an increase in urine production during the time period 01.00-04.00 (83 +/- 24 vs 52 +/- 18 and 45 +/- 22 ml, respectively, p < 0.05). At the end of the 6-month period of DDAVP treatment, the percentage response was similar between the ADH- and ADH+ enuretics (79% vs 75%). However, the time taken to achieve a response was quicker in the ADH- subjects. These data suggest the existence of a subgroup of enuretics whose underlying pathophysiology is the development of nocturnal polyuria probably due to a relative night-time ADH deficiency. Nocturnal sequential monitoring of urinary osmolality, as described above, allows identification of this subgroup.     

Effect of recombinant human basic fibroblast growth factor on angiogenesis during mandible fracture healing in rabbits

Objective: To investigate the effect of recombinant human basic fibroblast growth factor ( rhbFGF) on angiogenesis during mandible fracture healing in rabbit.Methods: Fifty adult white rabbits were used for animal model and randomly divided into a control group (25 rabbits) and an experimental group (25 rabbits). The membranous complex of rhbFGF and bovine type I collagen was prepared and implanted into the rabbit mandible fracture site under periosteum. The animals were sacrificed on 7, 14, 28, 56 and 84 days respectively after operation and the whole mandibles were harvested. The expression of factor Vm related antigen (F8-RA) in callus was examined with immimohistochemical staining.Results: The amounts of microvascular formation in calluses in the rhbFGF-treating group on days 7, 14, 28 and 56 were more than those of the control group (P < 0.01).Conclusions: The results indicated that rhbFGF could stimulate microvascular formation during mandible fracture healing in rabbits.     

Variation in urine composition in the human urinary tract: evidence of urothelial function in situ?

PURPOSE: An increased awareness of the concept that the urothelium has a significant transport function led us to question whether urine composition changes as it passes along the human lower urinary tract. MATERIALS AND METHODS: Urine samples from the bladder and renal pelvis were collected from 30 adults who underwent percutaneous nephrolithotomy (27) or ureteral stent insertion before lithotripsy (3). Urine was obtained from the 2 renal pelves (operative and contralateral sides) in 6 patients (24%). Urine pH was measured using an ultra-thin glass pH electrode. Urinary osmolality, Na and K were measured by micro-osmometry and flame photometry, respectively. Comparison of data sets was achieved using conventional nonparametric statistical methods. RESULTS: Median bladder urine pH in 30 patients, osmolality in 16, Na in 16 and K in 15 were significantly higher than in the renal pelvis at 6.76 (IQR 6.23 to 6.99), 469 mOsm. kg.1 (IQR 349 to 553), 132 (IQR 100 to 154) and 45 mM. (IQR 30 to 64) versus 6.08 (IQR 5.84 to 6.89), 308 mOsm. kg.1 (IQR 248 to 465), 90 (IQR 69 to 115) and 17 mM. (IQR 10 to 47), respectively (p < or = 0.05). There was no significant difference in these parameters in the urine of the paired renal pelves. CONCLUSIONS: Bladder urine pH, osmolality, Na and K significantly differ from values in the renal pelvis in moderately hydrated humans. Our data show that urine composition is modified in the lower urinary tract, supporting the concept of a dynamic urothelium. We propose that urothelial-urinary interactions and urinalysis need reappraisal, particularly in investigations of urinary stone formation and sensory bladder function.     

Comparison of the effects of human β-defensin 3, vancomycin, and clindamycin on Staphylococcus aureus biofilm formation

Despite improvements in surgical techniques and implant design in orthopedic surgery, implantation-associated infections are still a challenging problem for surgeons. In 2006, trace quantities of human β-defensin 3 (hBD-3) were found in human bone tissue and bone cells. Human β-defensin 3 is a 45-amino-acid peptide that is considered the most promising class of defensin antimicrobial peptides and may help in the prevention and treatment of implantation-associated infections. Studies of the effectiveness of hBD-3 against Staphylococcus aureus showed that hBD-3 was more potent at low concentrations than other antibiotics. The effect of hBD-3 on S aureus biofilms has not been reported. We studied the effect of hBD-3, vancomycin, and clindamycin on S aureus biofilms and on the survival of the bacteria in the biofilms.Staphylococcus aureus biofilms were examined with confocal scanning laser microscopy. Staining with LIVE/DEAD BacLight viability stain (Molecular Probes Europe BV, Leiden, The Netherlands) differentiated between live and dead bacteria within the biofilms, and extracellular polymeric substances (slime) from the biofilms was evaluated after staining with calcofluor white (Sigma Chemical Company, Rocky Hill, New Jersey). Human β-defensin 3 and clindamycin reduced the S aureus biofilm area. Human β-defensin 3 was significantly more effective against bacteria from the S aureus biofilms than was clindamycin. Vancomycin did not reduce the S aureus biofilm area.     

Protective Effects of GLP-1 on Glomerular Endothelium and Its Inhibition by PKCβ Activation in Diabetes

To characterize glucagon-like peptide (GLP)-1 signaling and its effect on renal endothelial dysfunction and glomerulopathy. We studied the expression and signaling of GLP-1 receptor (GLP-1R) on glomerular endothelial cells and the novel finding of protein kinase A–dependent phosphorylation of c-Raf at Ser259 and its inhibition of angiotensin II (Ang II) phospho–c-Raf(Ser338) and Erk1/2 phosphorylation. Mice overexpressing protein kinase C (PKC)β2 in endothelial cells (EC-PKCβ2Tg) were established. Ang II and GLP-1 actions in glomerular endothelial cells were analyzed with small interfering RNA of GLP-1R. PKCβ isoform activation induced by diabetes decreased GLP-1R expression and protective action on the renal endothelium by increasing its degradation via ubiquitination and enhancing phospho–c-Raf(Ser338) and Ang II activation of phospho-Erk1/2. EC-PKCβ2Tg mice exhibited decreased GLP-1R expression and increased phospho–c-Raf(Ser338), leading to enhanced effects of Ang II. Diabetic EC-PKCβ2Tg mice exhibited greater loss of endothelial GLP-1R expression and exendin-4–protective actions and exhibited more albuminuria and mesangial expansion than diabetic controls. These results showed that the renal protective effects of GLP-1 were mediated via the inhibition of Ang II actions on cRaf(Ser259) and diminished by diabetes because of PKCβ activation and the increased degradation of GLP-1R in the glomerular endothelial cells.Endothelial pathologies such as thrombotic microangiopathy and mesangiolysis are parts of glomerulopathy because of insulin resistance and diabetes, which are leading causes of clinical renal disease (1,2). Endothelial dysfunction is postulated to accelerate the progression of diabetic glomerulopathy as a result of the inhibition of endothelial nitric oxide (NO) synthesis (eNOS) and its product, NO (3).We have reported that activation of the β isoform of protein kinase C (PKC) by hyperglycemia can cause glomerular endothelial dysfunction and reduce eNOS activation partially owing to inhibition of insulin action on glomerular endothelial cells (4,5). Clinically, ruboxistaurin (RBX), a specific inhibitor of PKCβ, has been reported to improve endothelial dysfunction induced by hyperglycemia (4,6). Further, studies have associated PKCβ activation with glomerular pathology induced by hyperglycemia possibly due to the enhancement of angiotensin action (7). However, the biochemical mechanism by which PKCβ enhances angiotensin II (Ang II) action to accelerate the progression of diabetic glomerulopathy has not been clarified.Recently, glucagon-like peptide-1 (GLP-1) has been reported to biologically improve endothelial function and prevent some renal pathologies in diabetic rodents (8,9). However, a mechanistic explanation regarding GLP-1–protective action on the endothelial cell is unknown.GLP-1 is a gut incretin hormone that augments glucose-dependent insulin responses in the β cells (10). GLP-1 receptor (GLP-1R) is present abundantly in the gastrointestinal tract but has also been reported in endothelium and kidney and may stimulate NO production (8,11,12). In this study, we have identified a new biochemical mechanism for GLP-1 to inhibit Ang II inflammatory action via the c-Raf/extracellular signal–related kinase (Erk)1/2/plasminogen activator inhibitor (PAI)-1 pathway in glomerular endothelial cells. Further, we have demonstrated a dual signaling mechanism by which diabetes, via PKCβ activation, can increase Ang II action by increasing the inflammatory cytokines and extracellular matrix and inhibiting GLP-1–protective effects by reducing GLP-1R expression in the glomerular endothelium.     

The cryosurgical ablation of bone tissue by means of a new miniature cryoprobe – evaluation of the probe and adaption of the method to in vitro human bone

Until now, modern miniature cryoprobes have been used successfully for the local destruction of soft-tissue tumors without damaging adjacent healthy tissue. In this study, the methodology of cryoablation was applied to bone, and the freezing effect as well as the cooling capacity of the probe were examined. Freezing was performed by cooling one or two probes, with a diameter of 3.2 mm, to -180 degrees C with liquid nitrogen. The cooling capacity of the probes was determined under optic and thermic control in a homogenous reference gel (gelatin), followed by an in vitro measurement on human bone. The simultaneous use of 2 probes resulted in a synergistic effect which produced an almost spherical expansion of frozen area in the homogenous gelatin. In vitro freezing of human tibiae produced equivalent freezing temperatures, with one or two probes, in comparison to the homogenous gelatin. An adequate tissue cooling of bone matrix can be achieved through the use of one or more miniature cryoprobes so that after in vivo testing, the use of this probe could possibly become an alternative or supplement to the surgical resection of pathologic bone processes.     

Are true-negative nitrite results affected by a two-hour delay in dipstick/pad analysis of urine in incontinence pads?

OBJECTIVES: To determine if true-negative nitrite results of a urine dipstick pressed into an incontinence pad (dipstick/pad method) are affected by a 2-hour delay in analysis. DESIGN: A quantitative study. SETTING AND SUBJECTS: Clean-catch urine specimens from a convenience sample of clinic patients, staff, and long-term care facility residents. INSTRUMENT: Changes from negative to positive for each group of urine specimens were evaluated using percentages within the groups. METHODS: Urine specimens were collected and a portion was cultured. Urine specimens negative for a urinary tract infection were included in the study. A portion of the specimen was poured into an incontinence pad. Initial nitrite results were determined using a dipstick pressed into an incontinence pad. Pads with true-negative dipstick/pad nitrite results were tested 2 hours later in the same manner. Urine culture results determined groups: mixed colonies; 50,000 to 75,000 colony-forming units per milliliter of a single type of uropathogen; greater than or equal to 50,000 cfu/mL diphtheroids; no significant growth; and no growth. RESULTS: Of the 443 urine cultures negative for a urinary tract infection, 441 initial dipstick/pad nitrite results were negative. Two initial true-negative nitrite dipstick/pad results, or 0.5%, changed from negative to positive over the 2-hour period: 1 in the "mixed colonies" group (0.4%) and 1 in the "no growth" group (0.7%). CONCLUSIONS: Results of this study indicate that true-negative nitrite results of a dipstick pressed into urine in an incontinence pad do not appear to be affected by a 2-hour delay in analysis.     

Auxiliary liver transplantation in acute liver failure in the rat – an illustrated description of a new surgical approach

INTRODUCTION: To investigate auxiliary liver transplantation successfully in rats suffering from acute liver failure, we developed a new surgical approach. METHODS: A 70% hepatectomized liver graft was implanted into the right upper quadrant of the abdomen. The donor portal vein was anastomosed with the recipient's right renal artery using the splint technique. The donor infrahepatic vena cava was attached onto the recipient vena cava end to side. The bile duct was implanted into the duodenum.     

Effects of valganciclovir on spermatogenesis in renal transplant patients – results of a multicenter prospective nonrandomized study

Ganciclovir (GCV) inhibits spermatogenesis in preclinical studies but long-term effects on fertility in renal transplant patients are unknown. In a prospective, multicenter, open-label, nonrandomized study, male patients were assigned to Cohort A [valganciclovir (VGCV), a prodrug of GCV] (n = 38) or B (no VGCV) (n = 21) by cytomegalovirus prophylaxis requirement. Changes in semen parameters and DNA fragmentation were assessed via a mixed-effects linear regression model accounting for baseline differences. Sperm concentration increased post-transplant, but between baseline and treatment end (mean 164 days Cohort A, 211 days Cohort B), the model-based change was lower in Cohort A (difference: 43.82 × 10⁶/ml; P = 0.0038). Post-treatment, sperm concentration increased in Cohort A so that by end of follow-up (6 months post-treatment) changes were comparable between cohorts (difference: 2.09 × 10⁶/ml; P = 0.92). Most patients’ sperm concentration improved by end of follow-up; none with normal baseline concentrations (≥20 × 10⁶/ml) were abnormal at end of follow-up. Changes in seminal volume, sperm motility/morphology, DNA fragmentation, and hormone levels were comparable between cohorts at end of follow-up. Improvement in semen parameters after renal transplant was delayed in men receiving VCGV, but 6 months post-treatment parameters were comparable between cohorts.     

Internal fixation of acetabular fractures in an older population using the TIMI approach – Midterm results of a prospective study

IntroductionThe incidence of geriatric acetabular fractures continues to increase due to demographic changes. In the elderly, anterior column fractures are common, and standard approaches are associated with a considerable risk for surgery-associated complications. Therefore, a minimally invasive approach was developed in our department. The aim of this study was to examine early and mid-term results regarding the use of this novel two-incision minimally invasive (TIMI) approach in patients aged over 55 years with acetabular fractures.MethodsFrom July 2007 to April 2014, 47 patients aged over 55 years were treated via the TIMI approach; these patients were included in the present prospective study. The patients' characteristics, data, and early phase of care were assessed during acute care. A radiological evaluation comprised pre- and postoperative CT scans and x-rays, including Judet views at follow-up. Follow-up examinations were performed after 6 and 24 months and comprised a clinical and radiological examination and an evaluation of hip function (Harris Hip Score) and health-related quality of life (EQ-5D).ResultsThe mean age of the patients was 74 ± 11 years, with a gender ratio of 35/12 (m/f). The average operation time was 93 ± 30 min, and perioperative blood loss amounted to 858 ± 463 ml. In total, five (11%) complications associated with the operative procedure occurred, and revision surgery was necessary in three patients. We observed no wound infections, abdominal wall hernias or cases of heterotopic ossification in our sample. The Harris Hip Score at six months after surgery was 81, and it slightly improved to 84 after 24 months. The mean EQ5D index was 0.91 at six months after surgery and 0.92 at 24 months after surgery.ConclusionThe TIMI approach represents a valuable alternative to the ilioinguinal and modified Stoppa approach for the treatment of acetabular fractures located in the anterior column, which are often observed in geriatric patients.Level of evidenceTherapeutic Level II (Prospective cohort study).     

Is impaired immunity a consequence of surgery in patients infected by the human immunodeficiency virus?

BACKGROUND: Surgery affects immune function adversely in a variety of clinical settings. To date, there are no data assessing immune function in patients infected with the human immunodeficiency virus (HIV) who have had surgery. METHODS: A retrospective review was performed of 67 patients, of whom 46% were female, who underwent surgery while being treated for HIV infection. These patients were identified from a database collected over a ten-year period. The CD4(+) cell counts were analyzed according to the degree of immunosuppression (> or =500, 200-499, and <200 cells/mm(3), respectively). Viral titers also were assessed. RESULTS: Of the 17 patients with CD4(+) cell counts >500/mm(3) prior to surgery, 64.7% had unchanged counts after surgery (95% confidence interval [CI] 32.9%, 81.6%), whereas 35.2% of patients had lower CD4(+) counts after surgery (95% CI 14.2%, 61.7%). In patients with preoperative CD4(+) counts between 200 and 500/mm(3), 9.7% (95% CI 2.0%, 25.8%) had their counts decrease to <200 cells/mm(3), whereas in 29% (95% CI 14.2%, 48.0%) of patients, the counts increased to within the normal range. In the most immunosuppressed group (CD4(+) counts <200/mm(3)), 15.8% of patients (95% CI 3.4%, 39.6%) had their CD4(+) counts increase to the intermediate range. In the majority of patients, the viral titers remained unchanged, whereas 18.8% (n = 6) (95% CI 7.2%, 36.4%) had a decline in their titers. CONCLUSIONS: Surgery does not affect immune function adversely in HIV-infected patients, as judged by CD4(+) cell counts or viral titers.     

Clinical evaluation of recombinant human platelet – derived growth factor for the treatment of lower extremity diabetic ulcers

Purpose: The purpose of this study was to investigate the efficacy and safety of recombinant human platelet – derived growth factor (rhPDGF-BB) in a double-blind, placebo-controlled, multicenter study of patients with chronic diabetic ulcers.Methods: Patients with chronic, full-thickness, lower-extremity diabetic neurotrophic ulcers of at least 8 weeks' duration, free of necrotic and infected tissue after debridement, and with transcutaneous oxygen tensions of 30 mm Hg or greater were studied. A total of 118 patients were randomized to receive either topical rhPDGF-BB (2.2 μg/cm ² of ulcer area) or placebo until the ulcer was completely resurfaced or for a maximum of 20 weeks, whichever occurred first.Results: Twenty-nine (48%) of 61 patients randomized to the rhPDGF-BB group achieved complete wound healing during the study compared with only 14 (25%) of 57 patients randomized to the placebo group ( p = 0.01). The median reduction in wound area in the group given rhPDGF-BB was 98.8% compared with 82.1% in the group given placebo ( p = 0.09). There were no significant differences in the incidence or severity of adverse events between the rhPDGF-BB and placebo groups.Conclusions: Once-daily topical application of rhPDGF-BB is safe and effective in stimulating the healing of chronic, full-thickness, lower-extremity diabetic neurotrophic ulcers. (J VASC SURG 1995;21:71-81.)