Pranav Sinha

Comparison of 10 serum bone turnover markers in prostate carcinoma patients with bone metastatic spread: Diagnostic and prognostic implications

Our aim was to assess the diagnostic accuracy of bone markers in serum of patients with prostate cancer (PCa) for early detection of bone metastases and their usefulness as predictors of PCa‐caused mortality. In sera of 117 PCa patients (pN0M0, n = 39; pN1M0, n = 34; M1, n = 44), 35 healthy men and 35 patients with benign prostatic hyperplasia, bone formation markers [total and bone‐specific alkaline phosphatase (tALP, bALP), amino‐terminal procollagen propeptides of type I collagen (P1NP), osteocalcin (OC)], bone resorption markers [bone sialoprotein (BSP), cross‐linked C‐terminal (CTX) and cross‐linked N‐terminal (NTX) telopeptides of type I collagen, tartrate‐resistant acid phosphatase isoenzyme 5b (TRAP)] and osteoclastogenesis markers [osteoprotegerin (OPG), receptor activator of nuclear factor κB ligand (RANKL)] were measured. tALP, bALP, BSP, P1NP, TRAP, NTX and OPG were significantly increased in PCa patients with bone metastases compared to patients without metastases. OPG showed the best discriminatory power to differentiate between these patients. Logistic regression analysis resulted in a model with OPG and TRAP as variables that predicted bone metastasis with an overall correct classification of 93%. Patients with concentrations of OPG, P1NP, tALP, bALP, BSP, NTX, TRAP and CTX above cut‐off levels showed significantly shorter survival than patients with low marker concentrations. Multivariate Cox proportional hazards regression revealed that only OPG and BSP were independent prognostic factors for PCa‐related death. Thus, the importance of serum OPG in detecting bone metastatic spread, alone or in combination with other bone markers, and predicting survival in PCa patients has been clearly demonstrated.

A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis.

We report on a new silver stain especially developed for staining large gels (25 cm× 20 cm) from the Hoefer ISO‐DALT system for matrix‐assisted laser desorption/ ionization‐time of flight (MALDI‐TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI‐TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch‐to‐batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI‐TOF can be performed with protein loads as little as 100 νg per gel.

Increased expression of epidermal fatty acid binding protein, cofilin, and 14‐3‐3‐σ (stratifin) detected by two‐dimensional gel electrophoresis, mass spectrometry and microsequencing of drug‐resistant human adenocarcinoma of the pancreas

In order to study possible mechanisms leading to chemoresistance in pancreatic adenocarcinoma we examined the global protein expression of pancreatic cancer cells in vitro. We used a cell culture model derived from the adenocarcinoma of the pancreas (EPP85‐181P). A classical multidrug‐resistant subline, EPP85‐181RDB, selected in presence of daunorubicin, and an atypical multidrug‐resistant cell variant, EPP85‐181RNOV, selected in presence of mitoxantrone, were analyzed using two‐dimensional electrophoresis. After staining and image analysis, spots of interest were isolated using preparative two‐dimensional electrophoresis and subjected to mass spectrometry and microsequencing. Three proteins, E‐FABP, cofilin, and 14‐3‐3‐σ (stratifin), were overexpressed in chemoresistant cell lines. Cofilin was present in both multidrug in chemoresistant cell lines. Cofilin was present in both multidrug‐resistant cell lines. E‐FABP and 14‐3‐3‐σ (stratifin) was found to be overexpressed only in the mitoxantrone‐selected atypical multidrug‐resistant cell line. The possible significance of these findings is discussed.

Altered cell-mediated immunity and increased postoperative infection rate in long-term alcoholic patients.

Background: Preoperative alteration of T cell–mediated immunity as well as an altered immune response to surgical stress were found in long-term alcoholic patients. The aim of this study was to evaluate perioperative T cell–mediated immune parameters as well as cytokine release from whole blood cells after lipopolysaccharide stimulation and its association with postoperative infections. Methods: Fifty-four patients undergoing elective surgery of the aerodigestive tract were included in this prospective observational study. Long-term alcoholic patients (n = 31) were defined as having a daily ethanol consumption of at least 60 g and fulfilling the Diagnostic and Statistical Manual of Mental Disorders for either alcohol abuse or alcohol dependence. The nonalcoholic patients (n = 23) were defined as drinking less than 60 g ethanol/day. Blood samples to analyze the immune status were obtained on morning before surgery and on the morning of days 1, 3, and 5 after surgery. Results: Basic patient characteristics did not differ between groups. Before surgery, the T helper 1:T helper 2 ratio (Th1: Th2) was significantly lower (P < 0.01), whereas plasma interleukin 1β and lipopolysaccharide-stimulated interleukin 1ra from whole blood cells were increased in long-term alcoholic patients. After surgery, a significant suppression of the cytotoxic lymphocyte ratio (Tc1:Tc2), the interferon γ:interleukin 10 ratio from lipopolysaccharide-stimulated whole blood cells, and a significant increase of plasma interleukin 10 was observed. Long-term alcoholics had more frequent postoperative infections compared with nonalcoholic patients (54%vs. 26%; P = 0.03). Conclusions: T helper cell–mediated immunity was significantly suppressed before surgery and possibly led to inadequate cytotoxic lymphocyte and whole blood cell response in long-term alcoholic patients after surgery. This altered cell-mediated immunity might have accounted for the increased infection rate in long-term alcoholic patients after surgery.

Identification of novel proteins associated with the development of chemoresistance in malignant melanoma using two-dimensional electrophoresis.

A model system for studying chemoresistance in human melanoma cells (MeWo) has been established utilizing the four commonly used cytotoxic drugs vindesine, cisplatin, fotemustine and etoposide to yield stable drug‐resistant sublines. We analyzed phenotypical differences between MeWo cells and their chemoresistant counterparts using two‐dimensional electrophoresis. Proteins that were overexpressed in chemoresistant cell lines were purified and identified using matrix assisted laser desorption/ionization‐time of flight — mass spectrometry (MALDI‐TOF‐MS) and microsequencing. Here we show that four proteins, namely the translationally controlled tumor protein, the human elongation factor 1‐δ, tetratricopeptide repeat protein and the isoform 14‐3‐3‐γ of the 14‐3‐3‐family are overexpressed in chemoresistant melanoma cell lines. The significance of these findings is now being verified using transfection experiments with the aim of developing more effective chemotherapy protocols.

The role of automated urine particle flow cytometry in clinical practice

Urine particle flow cytometers (UFC) have improved count precision and accuracy compared to visual microscopy and offer significant labor saving. The absence of an internationally recognized reference measurement procedure, however, is a serious drawback to their validation. Chamber counting by phase contrast microscopy of supravitally-stained uncentrifuged urine is considered the best candidate for reference. The UF-100 (Sysmex Corporation, Japan) identifies RBC, WBC, squamous epithelial cells, transitional epithelial and renal tubular cells (SRC), bacteria, hyaline and inclusional casts, yeast-like cells, crystals and spermatozoa, using argon laser flow cytometry. Evaluations have established acceptable linearity over useful working ranges, with an imprecision that is consistently and significantly less than microscopy, and with negligible carry-over. Comparisons of UFC with chamber counts, quantitative urine microscopy, sediment counts, test strips, bacterial culture and urine density are reviewed. Clinical studies include diagnosis and monitoring of urinary tract infection; localization of the sites of hematuria; and diagnosis, monitoring and exclusion of renal disease. The most popular approach is to combine test strips with UFC for primary screening either always by both methods or by using test strips for analytes unrelated to particles analyzed by UFC. Expert systems now exist combining both test modalities based on user definable decision rules. The implementation of such a strategy significantly reduces microscopy review and saves time and expense without diminishing clinical utility.

Alcohol withdrawal severity is decreased by symptom-orientated adjusted bolus therapy in the ICU

ObjectiveTo examine the effect of bolus vs. continuous infusion adjustment on severity and duration of alcohol withdrawal syndrome (AWS), the medication requirements for AWS treatment, and the effect on ICU stay in surgical intensive care unit (ICU) patients.Design and settingProspective randomized, double-blind controlled trial in a surgical ICU.Patients44 patients who developed AWS after admission to the ICU.InterventionsPatients were randomized to either (a) a continuous infusion course of intravenous flunitrazepam (agitation), intravenous clonidine (sympathetic hyperactivity), and intravenous haloperidol (productive psychotic symptoms) if needed (infusion-titrated group), or (b) the same medication (flunitrazepam, clonidine, or haloperidol) bolus adjusted in response to the development of the signs and symptoms of AWS (bolus-titrated group).Measurements and resultsThe administration of “as-needed” medication was determined using a validated measure of the severity of AWS (Clinical Institute of Withdrawal Assessment). Although the severity of AWS did not differ between groups initially, it significantly worsened over time in the infusion-titrated group. This required a higher amount of flunitrazepam, clonidine, and haloperidol. ICU treatment was significantly shorter in the bolus-titrated group (median difference 6 days) due to a lower incidence of pneumonia (26% vs. 43%).ConclusionsWe conclude that symptom-orientated bolus-titrated therapy decreases the severity and duration of AWS and of medication requirements, with clinically relevant benefits such as fewer days of ventilation, lower incidence of pneumonia, and shorter ICU stay.

Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells

The therapy of advanced cancer using chemotherapy alone or in combination with radiation or hyperthermia yields an overall response rate of about 20-50%. This success is often marred by the development of resistance to cytostatic drugs. Our aim was to study the global analysis of protein expression in the development of chemoresistance in vitro. We therefore used a cell culture model derived from the gastric carcinoma cell line EPG 85-257P. A classical multidrug-resistant subline EPG85-257RDB selected to daunorubicin and an atypical multidrug-resistant cell variant EPG85-257RNOV selected to mitoxantrone, were analysed using two-dimensional electrophoresis in immobilized pH-gradients (pH 4.0-8.0) in the first dimension and linear polyacrylamide gels (12%) in the second dimension. After staining with coomassie brilliant blue, image analysis was performed using the PDQuest system. Spots of interest were isolated using preparative two-dimensional electrophoresis and subjected to microsequencing. A total of 241 spots from the EPG85-257RDB-standard and 289 spots from the EPG85-257RNOV-standard could be matched to the EPG85-257P-standard. Microsequencing after enzymatic hydrolysis in gel, mass spectrometric data and sequencing of the peptides after their fractionation using microbore HPLC identified that two proteins annexin I and thioredoxin were overexpressed in chemoresistant cell lines. Annexin I was present in both the classical and the atypical multidrug-resistant cells. Thioredoxin was found to be overexpressed only in the atypical multidrug-resistant cell line.

Nitroglycerin to facilitate fetal extraction during cesarean delivery.

Objective To determine the long-term outcomes of children exposed in utero to maternal parvovirus B19 infection. Methods All pregnant women with serologic evidence of recent parvovirus B19 infection and a comparison group with serologic evidence of past infection from January 1988 to December 1994 were sent questionnaires or contacted by phone about the health and development of their children. Information requested included: pregnancy complications, date of delivery, birth weight, sex, birth defects, need for special care, significant health problems, and developmental delays. All women had serology done at either the Centers for Disease Control and Prevention or the virology laboratory of the Connecticut Department of Health. The data were analyzed using descriptive statistics, χ2 analysis with Fisher exact test, or Student t test in appropriate cases. P < .05 was considered significant. Results Outcome information was obtained from 113 of 117 immunoglobulin-M positive women. The 113 respon-dents had 103 term singletons, two sets of twins (of which one neonate died of complications of prematurity), one hydropic stillborn, four spontaneous abortions, and one ectopic pregnancy. The mean gestational age at time of exposure was 15.6 weeks. The median age of the liveborn infants in study and comparison groups was 4 years. Eight of the 108 (7.3%) surviving children, one set of twins (exposed at 27 weeks), and six singletons (exposed at 7, 8, 9, 20, 27, and 35 and 35 weeks) had developmental delays in speech, language, information processing, and attention. Outcomes were obtained for 99 of 110 patients with past infection; they had 83 liveborn singletons, five sets of twins, two stillborns, and five spontaneous abortions. Seven of the 93 (7.5%) children had developmental delays, similar to the study group. Post-hoc power analysis revealed that 712 infected patients would be needed to find a twofold difference in the risk of abnormal neurologic development; our study had 30% power to find such a difference. Conclusion There is no apparent increase in the frequency of developmental delays in children with exposure in utero to parvovirus, but larger studies are needed.

Bone resorption parameters [carboxy-terminal telopeptide of type-I collagen (ICTP), amino-terminal collagen type-I telopeptide (NTx), and deoxypyridinoline (Dpd)] in MGUS and multiple myeloma.

Abstract: Skeletal morbidity is a major problem in multiple myeloma. Histomorphometric studies have demonstrated that increased bone resorption can be present even in the absence of radiographic abnormalities. To overcome diagnostic problems in estimating the activity of bone resorption, new laboratory parameters that reflect bone metabolism accurately are urgently needed. We analyzed three parameters of osteoclastic bone destruction, i.e. deoxypyridinoline (Dpd) and amino‐terminal collagen type‐I telopeptide (NTx) in urine and carboxy‐terminal telopeptide of type‐I collagen (ICTP) in serum, of 75 patients with multiple myeloma (n = 57) or monoclonal gammopathy of undetermined significance (MGUS, n = 18) by ELISA/RIA techniques. Serum ICTP and urinary Dpd levels increased parallel to the stage of the disease and differed significantly (P < 0.001 for ICTP and P = 0.03 for Dpd) between MGUS, myeloma stage I, and myeloma in stages II and III according to Salmon and Durie. ICTP and Dpd were significantly elevated in patients with multiple myeloma in stage I compared to individuals with MGUS, while no significant difference was found for NTx. In this first study comparing the prognostic relevance of ICTP, NTx, and Dpd in multiple myeloma patients, ICTP was found to be a prognostic factor for overall survival in the Kaplan–Meier analysis (log‐rank test: P < 0.03). Urinary NTx showed borderline significance (P = 0.05), and Dpd had no prognostic value in the survival analysis. Our data show that serum ICTP and urinary Dpd levels increase in parallel to advanced disease stages, and gives the first report on a significant difference in the bone resorption parameters ICTP and Dpd between individuals with MGUS and patients with myeloma in stage I. Among the bone resorption parameters studied serum ICTP was found to be the best prognostic factor for survival in multiple myeloma.