Synchrotron infrared spectromicroscopy as a novel bioanalytical microprobe for individual living cells: cytotoxicity considerations

Synchrotron radiation-based Fourier transform infrared spectromicroscopy is a newly emerging analytical tool capable of monitoring the biochemistry within an individual living mammalian cell in real time. This unique technique provides infrared (IR) spectra, hence chemical information, with high signal to noise at spatial resolutions as fine as 3-10 microm. Mid-IR photons are too low in energy (0.05-0.5 eV) to either break bonds or to cause ionization, and the synchrotron IR beam has been shown to produce minimal sample heating. However, an important question remains, "Does the intense synchrotron beam induce any cytotoxic effects in living cells?" In this work, we present the results from a series of standard biological assays to evaluate any short- and/or long-term effects on cells exposed to the synchrotron radiation-based infrared (SR-IR) beam. Cell viability was tested using alcian blue dye exclusion and colony formation assays. Cell-cycle progression was tested with bromodeoxyuridine (BrdU) uptake during DNA synthesis. Cell metabolism was tested using a 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. All control, 5, 10, and 20 min SR-IR exposure tests (267 total and over 1000 controls) show no evidence of cytotoxic effects. Concurrent infrared spectra obtained with each experiment confirm no detectable biochemical changes between control and exposed cells.     

UKPDS 59: hyperglycemia and other potentially modifiable risk factors for peripheral vascular disease in type 2 diabetes

OBJECTIVE: To determine the role of hyperglycemia in prospective analyses of peripheral vascular disease (PVD) in type 2 diabetes, taking into account other potential risk factors. RESEARCH DESIGN AND METHODS: Potential risk factors for the development of PVD were examined in 3,834 of 5,102 individuals enrolled in the U.K. Prospective Diabetes Study (UKPDS) without PVD at diagnosis of diabetes, followed for 6 years, and for whom relevant data were available. PVD was defined as two of the following: ankle-arm blood pressure index < 0.8, absence of both dorsalis pedis and posterior tibial pulses to palpation in one or both legs, and intermittent claudication. Logistic regression was used to estimate the association between potential risk factors measured 3-4 months after diagnosis of diabetes and incident PVD. The prevalence of PVD at 3-year intervals to 18 years was determined. RESULTS: Hyperglycemia, assessed as HbA(1c), was associated with an increased risk for incident PVD, independent of other risk factors including age, increased systolic blood pressure, reduced HDL cholesterol, smoking, prior cardiovascular disease, peripheral sensory neuropathy, and retinopathy. Each 1% increase in HbA(1c) was associated with a 28% increased risk of PVD (95% CI 12-46), and each 10-mmHg increase in systolic blood pressure with a 25% increase in risk (95% CI 10-43). CONCLUSIONS: Hyperglycemia, as well as smoking, dyslipidemia, and blood pressure are potentially modifiable risk factors for the development of PVD.     

Development and progression of nephropathy in type 2 diabetes: the United Kingdom Prospective Diabetes Study (UKPDS 64)

BACKGROUND: The progression of nephropathy from diagnosis of type 2 diabetes has not been well described from a single population. This study sought to describe the development and progression through the stages of microalbuminuria, macroalbuminuria, persistently elevated plasma creatinine or renal replacement therapy (RRT), and death. METHODS: Using observed and modeled data from 5097 subjects in the UK Prospective Diabetes Study, we measured the annual probability of transition from stage to stage (incidence), prevalence, cumulative incidence, ten-year survival, median duration per stage, and risk of death from all-causes or cardiovascular disease. RESULTS: From diagnosis of diabetes, progression to microalbuminuria occurred at 2.0% per year, from microalbuminuria to macroalbuminuria at 2.8% per year, and from macroalbuminuria to elevated plasma creatinine (>or=175 micromol/L) or renal replacement therapy at 2.3% per year. Ten years following diagnosis of diabetes, the prevalence of microalbuminuria was 24.9%, of macroalbuminuria was 5.3%, and of elevated plasma creatinine or RRT was 0.8%. Patients with elevated plasma creatinine or RRT had an annual death rate of 19.2% (95% confidence interval, CI, 14.0 to 24.4%). There was a trend for increasing risk of cardiovascular death with increasing nephropathy (P < 0.0001), with an annual rate of 0.7% for subjects in the stage of no nephropathy, 2.0% for those with microalbuminuria, 3.5% for those with macroalbuminuria, and 12.1% with elevated plasma creatinine or RRT. Individuals with macroalbuminuria were more likely to die in any year than to develop renal failure. CONCLUSIONS: The proportion of patients with type 2 diabetes who develop microalbuminuria is substantial with one quarter affected by 10 years from diagnosis. Relatively fewer patients develop macroalbuminuria, but in those who do, the death rate exceeds the rate of progression to worse nephropathy.     

Modulation of mitochondrial adenosine triphosphate-sensitive potassium channels and sodium-hydrogen exchange provide additive protection from severe ischemia-reperfusion injury

BACKGROUND: Preconditioning and inhibition of sodium-proton exchange attenuate myocardial ischemia-reperfusion injury by means of independent mechanisms that might act additively when used together. The hypothesis of this study is that treatment with a sodium-proton exchange inhibitor and a mitochondrial adenosine triphosphate-sensitive potassium channel opener produces superior functional recovery and a greater decrease in left ventricular infarct size compared with treatment with either drug alone in a model of severe global ischemia. METHODS: Isolated crystalloid-perfused rat hearts (n = 8 hearts per group) were administered vehicle (control, 0.04% dimethyl sulfoxide), diazoxide (100 micromol/L in 0.04% dimethyl sulfoxide), cariporide (10 micromol /L in 0.04% dimethyl sulfoxide), or diazoxide and cariporide before 40 minutes of ischemia at 35.5 degrees C to 36.5 degrees C and 30 minutes of reperfusion. RESULTS: The combination group had superior postischemic systolic function compared with that seen in the cariporide, diazoxide, and control groups (recovery of developed pressure: 91% +/- 7% vs 26% +/- 5%, 35% +/- 6%, and 16% +/- 3%, respectively; P <.05). Postischemic diastolic function in the combination group was superior compared with that seen in the other groups (change(pre-post) diastolic pressure of 67 +/- 4 mm Hg with control, 49 +/- 11 mm Hg with diazoxide, 59 +/- 10 mm Hg with cariporide, and 3 +/- 3 mm Hg with diazoxide and cariporide combination; P <.05). The left ventricular infarct area was less in the combination group compared with that in the cariporide, diazoxide, and control groups (6% +/- 2% vs 35% +/- 7%, 25% +/- 3%, and 37% +/- 9%, respectively; P <.05). CONCLUSIONS: Combining a selective mitochondrial adenosine triphosphate-sensitive potassium channel opener with a selective reversible inhibitor of sarcolemmal sodium-proton exchange improves recovery of contractile function from severe global ischemia in the isolated buffer-perfused rat heart. The putative mechanism for this benefit is superior protection of mitochondrial function.     

Laparoscopic treatment of post renal transplant lymphoceles

Background: Traditionally, a post transplant lymphocele (PTL) is drained by widely opening the wall connecting the lymphocele cavity to the intraperitoneal space via laparotomy. We hypothesize that laparoscopic techniques can be effectively used for the treatment of PTL.Methods: Patients requiring intervention for PTL between 1993 and 2002 were identified via a retrospective review. Results of drainage via laparotomy and laparoscopy were compared.Results: During the study period 685 renal transplants (391 cadaveric, 294 living) were performed. The incidence of lymphocele was 5% [34/685 (36 cases)]. The indications for surgical drainage were local symptoms (69%), graft dysfunction (14%), or both (17%). The mean time to surgical therapy was 4.9 months. Laparoscopic drainage was performed in 25 patients (74%) and open drainage in 9 patients (26%). Open procedures were performed in cases for: previous abdominal surgery (5), undesirable lymphocele characteristics or location (2), or with concomitant open procedures (3). There were no conversions or operative complications in either group. There was no difference in operative time for the laparoscopic group vs the open group (108 ± 6 vs 123 ± 18 min, p = 0.8). Hospital stay was significantly shorter for the laparoscopic group (1.7 ± 0.8 vs 3.8 ± 1.0, p = 0.0007), with 88% of laparoscopic patients being either overnight admissions or same day surgery. Two patients (5%) developed symptomatic recurrences requiring reoperation [1 laparoscopic (4%), 1 open (10%)].Conclusions: Laparoscopic fenestration of a peritransplant lymphocele is a safe and effective treatment. The large majority of patients treated with laparoscopic fenestration were discharged within one day of surgery. Unless contraindications exist, laparoscopy should be considered first-line therapy for the surgical treatment of posttransplant lymphocele.     

Human parainfluenza virus-associated hospitalizations among children less than five years of age in the United States

BACKGROUND: Human parainfluenza viruses 1 through 3 (HPIV-1-3) are important causes of respiratory tract infections in young children. This study sought to provide current estimates of HPIV-1-3-associated hospitalizations among US children. METHODS: Hospitalizations for bronchiolitis, bronchitis, croup and pneumonia among children age <5 years were determined for the years 1979 through 1997 using the National Hospital Discharge Survey. Average annual hospitalizations during the last 4 years of the study for each of these four diseases were multiplied by the proportions of each disease associated with HPIV-1-3 infection (as previously reported in hospital-based studies) to estimate hospitalizations potentially associated with HPIV-1-3 infections. Seasonal trends in HPIV-1-3-associated hospitalizations were compared with HPIV detections in the National Respiratory and Enteric Virus Surveillance System, which prospectively monitors respiratory viral detections throughout the United States. RESULTS: The proportions of hospitalizations associated with HPIV infection for each disease varied widely in the 6 hospital-based studies we selected. Consequently our annual estimated rates of hospitalization were broad: HPIV-1, 0.32 to 1.59 per 1,000 children; HPIV-2, 0.10 to 0.86 per 1,000 children; and HPIV-3, 0.48 to 2.6 per 1,000 children. Based on these data HPIV-1 may account for 5,800 to 28,900 annual hospitalizations; HPIV-2 for 1,800 to 15,600 hospitalizations; and HPIV-3 for 8,700 to 52,000 hospitalizations. CONCLUSIONS: We provide broad, serotype-specific estimates of US childhood hospitalizations associated with HPIV infections. More precise estimates of HPIV-associated hospitalizations would require large prospective studies of HPIV-associated diseases by more sensitive viral testing methods, such as polymerase chain reaction techniques.