Possible proteomic biomarkers for the detection of pancreatic cancer in oral fluids.

The 80% mortality rate of pancreatic-cancer (PC) makes early diagnosis a challenge. Oral fluids (OF) may be considered the ultimate body fluid for non-invasive examinations. We have developed techniques to improve visualization of minor OF proteins thereby overcoming major barriers to using OF as a diagnostic fluid. The aim of this study was to establish a short discriminative panel of OF biomarkers for the detection of PC. Unstimulated OF were collected from PC patients and controls (n = 30). High-abundance-proteins were depleted and the remaining proteins were analyzed by two-dimensional-gel-electrophoresis and quantitative dimethylation-liquid-chromatography-tandem mass-spectrometry. Label-free quantitative-mass-spectrometry analysis (qMS) was performed on 20 individual samples (n = 20). More than 100 biomarker candidates were identified in OF samples, and 21 had a highly differential expression profile. qMS analysis yielded a ROC-plot AUC value of 0.91 with 90.0% sensitivity and specificity for a combination of five biomarker candidates. We found a combination of five biomarkers for PC. Most of these proteins are known to be related to PC or other gastric cancers, but have never been detected in OF. This study demonstrates the importance of novel OF depletion methodologies for increased protein visibility and highlights the clinical applicability of OF as a diagnostic fluid.

[Screening saliva].

Oral Fluids (OF) are a complex mixture including components deriving from, salivary glands, blood, nasal and bronchial secretions, mucosal lining cells and microbiota. Therefore, OF as a mirror of the body, were suggested as an important diagnostic fluid for the detection of both, oral and systemic diseases. OF as diagnostic fluids have several advantages; their collection is easy, inexpensive and noninvasive, they are suitable for home use and for epidemiology researches, they are easy to store and ship, do not clot and enable fast detection. OF based diagnostics research accomplished a great advance during the last decade. This is mainly due to biotechnology improvements such as 2-D Fluorescence Difference Gel Electrophoresis, quantitative Mass Spectrometry and bioinformatics systems. These technologies enabled the detection of more than 3000 proteins in oral fluids, as well as the establishment of a panel of biomarkers to different human pathological conditions (i.e. periodontitis, Sjögren's Syndrome, oral cancer, pancreatic cancer etc). However, this diagnostic field has several drawbacks, mainly due to oral fluids variance composition, blood contamination as a result of gingivitis or mucosal injuries, the lack of a single established collection protocol and the presence of high abundant components in OF. This article summarizes the current research, and provides an outlook toward the foundation of this unique body fluid as a major player in the diagnostic field.

Non-syndromic autosomal recessive congenital ichthyosis in the Israeli population.

BACKGROUND: Autosomal recessive congenital ichthyosis (ARCI) is the term given to a complex and heterogeneous group of cornification disorders associated with mutations in at least eight distinct genes. Mutation distribution and prevalence rates are instrumental for the design of diagnostic strategies in ARCI but have not yet been systematically explored in the Israeli population. Previous data suggest that the demographic features specific to Middle Eastern populations, such as a high frequency of consanguineous marriages, may have an effect on the molecular epidemiology of genodermatoses. METHODS: We systematically assessed all families with ARCI presenting at our clinics over a period of 9 years, using a combination of homozygosity mapping, direct sequencing and PCR-restriction fragment length polymorphism assays. RESULTS: In total, 20 families with ARCI were assessed, and causative mutations were identified in 7 genes: TGM1 (30% of patients), ALOX12B (20%), ABCA12 (5%), CYP4F22 (10%), ALOXE3 (10%), LIPN (5%) and NIPAL4 (5%) Two families (10%) had mutations mapped to an ARCI-associated locus on 12p11.2-q13, while no mutation was found for one additional kindred. In the subgroup of families of Arab Muslim origin, mutations were identified most frequently in ALOX12B and TGM1 (31%), whereas the other subgroups displayed a subtype distribution very similar to that previously reported in western populations. CONCLUSIONS: The present data point to the need for population-tailored mutation screening strategies in genetically heterogeneous genodermatoses, based on the relative prevalence of the disease subsets.

Sialochemistry and cortisol levels in patients with Sjogren's syndrome.

OBJECTIVES: (i) To determine whether salivary cortisol and electrolyte levels differ between patients with Sjogren's syndrome (SjS) and healthy individuals. (ii) To assess correlations between whole-saliva cortisol and some clinical manifestations in patients with SjS. METHODS: A total of 24 healthy women (mean age 49.3±9.8) served as controls (C) vis-à-vis 17 patients with SjS (mean age 55.5±15.7). Salivary cortisol concentration was determined, and sialochemistry analysis was performed. RESULTS: Significantly lower saliva flow rates and higher salivary chloride (Cl(-) ), potassium (K(+) ), and Ca(2+) levels were found in the SjS group. No significant differences or correlations were found in other parameters, including sodium (Na(+) ), magnesium (Mg(2+) ), phosphate ((-) ), urea (U), and salivary cortisol levels. CONCLUSION: Increased whole-salivary output of Cl(-) and K(+) in SjS may reflect release from apoptotic rests of acinar cells after secondary necrosis. Normal levels of salivary Na(+) , Mg(2+) , and (-) argue against concentration effect, deranged tubular function or cortisol (mineralocorticosteroid) effect as the cause for these findings. Increased salivary Ca(2+) levels probably reflect leakage of plasma Ca(2+) through the injured oral mucosa in SjS. In spite of disease-associated stress, salivary cortisol, a stress biomarker, was not increased, suggesting insufficient hypothalamus-pituitary-adrenal (HPA) axis response and/or local consumption of cortisol by lymphocyte infiltrates.

Improved visualization of low abundance oral fluid proteins after triple depletion of alpha amylase, albumin and IgG.

OBJECTIVES: The aim of this study was to examine whether triple depletion of salivary-α-amylase (sAA), albumin (Alb) and immunoglobulins G (IgGs) may improve the visualization capability of proteins in two-dimensional gel electrophoresis (2-DE) of oral fluids (OF). SUBJECTS AND METHODS: Oral fluids from healthy volunteers were subjected sequentially to sAA removing device followed by application to an Alb and IgG immunoaffinity column (triple depletion). The depleted OF samples were analyzed using SDS-PAGE followed by 2-DE and protein identification using ion-trap mass spectrometry (MS). RESULTS: This specific triple depletion technique unmasked spots never visualized before. A total of 36 new spots were observed after depletion (348 vs 312 before depletion). Moreover, 58 spots showed more than twofold increase intensity after depletion. In the 60-69kDa area, the depletion procedure unmasked 14 proteins including HSP70, LTA4H, L-Plastin, Desmoplakin that are known to be involved in disease pathogenesis. CONCLUSION: The ability to selectively remove and elute the most abundant OF proteins visualized on the 2-DE represents an important step in the characterization of human OF. The better visualization and gel resolution achieved will improve quantification abilities in 2-DE and in tag-MS leading to better identification of disease-specific biomarkers. We further analyzed the eluted Alb and IgGs isoforms suggesting a new methodology venue for OF.

Comparative proteomic analysis of human oral fluids according to gender and age.

BACKGROUND: Recently, interest in finding disease bio-markers in human body fluids including oral fluids (OF), mainly saliva has increased. However, the physiologic differences in salivary proteins according to gender and age should be explored to establish a clinical diagnostic tool. OBJECTIVE: To compare OF protein expression according to gender and age, using proteomic approaches. MATERIALS AND METHODS: Oral fluids from 27 healthy volunteers (14 males, 13 females) was collected and divided into three age-groups. OF proteins were separated by means of 2D-SDS-PAGE. A total of 51 proteins in 37 protein spots were identified by ESI-MS/MS. RESULTS: Gender differences revealed six proteins with significant higher expression in females, including ß-2-microglobulin and transferrin. Age differences revealed decrease in expression of eight proteins with aging among males and seven proteins differentially expressed with aging among females including prolactin inducible protein, Ig-k light chain, transferrin, and calgranulin-B. CONCLUSION: Proteomic analysis of OF revealed differences in protein expression according to gender and age and therefore can highlight future use of this technique for diagnostic purposes in health and in disease.

Different proteomic protein patterns in saliva of Sjögren's syndrome patients.

OBJECTIVE: To investigate the salivary protein profile in patients with Sjögren's syndrome (SS), and healthy control subjects. MATERIALS AND METHODS: Unstimulated whole saliva samples were collected from 16 age-matched females; eight healthy subjects and eight patients diagnosed with SS (six primary SS, one incomplete SS and one primary SS associated with B cell lymphoma). Proteins were extracted and separated individually by 2D sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Selected protein spots of interest were analysed by electrospray ionization--tandem mass spectrometry. Obtained data were searched against the Swiss-Prot and NCBI non-redundant protein databases using Mascot software. RESULTS: Two groups of patterns of protein expression were observed in the eight SS patients: a major group (six patients) with significant expression differences from the healthy subjects and the second group (two patients) with a pattern similar to the eight healthy subjects. CONCLUSION: In this preliminary study, protein expression differences were found between SS patients and healthy subjects. Individual analysis of SS patients exhibited two patterns of protein expression with no direct relation to the clinical, serological or histological severity of disease. This study emphasizes the difficulty of the present proteomic knowledge to diagnose and monitor the sequel of SS development.

Heart transplantation in a patient with multiple sclerosis and mitoxantrone-induced cardiomyopathy.

We describe a 30-year-old man with end-stage heart failure after therapy with mitoxantrone for multiple sclerosis. A successful orthotopic heart transplantation was performed when intensified medical therapy failed to improve the patient's hemodynamics. In spite of the severe underlying disease he did well on dual immunosuppression with methylprednisone and cyclosporine. Neurologic symptoms remained stable throughout the procedure and, after 2 months, he resumed preoperative ambulatory status. Eight years after the operation, the patient is now in New York Heart Association (NYHA) Class I status. Using canes, he is able to walk short distances. Repeated urinary tract infections caused by Escherichia coli became a problem, but have been controlled by long-term oral antibiotic prophylaxis with trimethoprim.

Optimization of the immunosuppressive protocol after lung transplantation.

BACKGROUND: The successful use of tacrolimus (Tac)-based immunosuppressive therapy in organ transplantation and our own positive experience in heart transplantation led us to investigate regimens including this agent at our center for lung transplantation. METHODS: From 1991 to 1998, 86 patients underwent lung transplants at our center and 78 of them were included in this analysis. The first 34 patients were treated with cyclosporin (CsA), azathioprine (Aza), and rabbit antilymphocyte globulin; the subsequent 30 patients received Tac with Aza, and the most recent 12 patients Tac with mycophenolate mofetil (MMF). In addition, all patients received prednisone. RESULTS: The number of acute rejection episodes per 100 patient days was 1.5, 0.6, and 0.3 for three treatment groups, respectively. Similarly, the incidence of refractory acute rejection per 100 patient days was lower in both Tac groups (0.20, 0.03, and 0, respectively). Freedom from acute rejection was highest in the Tac-MMF group (P=0.0037 vs. Tac/Aza, P=0.0007 vs. CsA/Aza). Freedom from recurrent acute rejection was significantly higher in both Tac groups (P=0.027 Tac/ Aza vs. CsA/Aza and P=0.025 Tac/MMF vs. CsA/Aza). The incidence of infections per 100 patient days was similar (0.8, 0.5, and 0.8) in all three treatment groups, with a similar distribution of fungal, bacterial, and viral infections. Freedom from infection also showed no difference. The survival rate was significantly higher for the Tac population, with actuarial 1- and 3-year survival rates of 93% and 71%, compared with the CsA group (71% and 51%, respectively, P=0.04). Prevalence of renal dysfunction (creatinine >2.0 mg/ dL) was 18%, 13%, and 0% in the 3 treatment groups, respectively. The development of glucose metabolism disorders was lower in the CsA group than in the Tac-Aza group (15% vs. 27%, P<0.05). CONCLUSIONS: Tac-based immunosuppressive therapy results in a lower rate of acute rejection after pulmonary transplantation, with similar infection rates and a slightly higher incidence of new onset diabetes mellitus compared with CsA-based therapy.

Classic and iatrogenic Kaposi's sarcoma. Histopathological patterns as related to clinical course.

The histological findings in the first diagnostic biopsy of 50 patients with Kaposi's sarcoma (KS) were classified and were related to the clinical course. Forty-one patients had the "classic KS," of whom 31 had a benign course and 10 an aggressive disease. Nine patients had iatrogenically induced KS. Histologically the following subtypes were found: mixed type in 18 patients, spindle type in 16, early type in 14, and hemangiomatous and lymphangiomatous types in one each. We did not find any statistical relationship between the histological subtypes, degree of nuclear atypia, number of mitoses, and density of the inflammatory cell infiltrates in the KS lesions, and the clinical types and course of the disease. The results of this study do not support the possibility of a prognostic importance regarding the histological features of the early lesions in patients with classic or iatrogenic Kaposi's sarcoma.

Mal de Meleda keratoderma with pseudoainhum.

Pseudoainhum is an infrequent complication in the autosomal-recessive keratodermas. We describe two related families in which the diagnosis of mal de Meleda keratoderma has been confirmed by mode of inheritance and ultrastructural findings. One family member, a 9-year-old girl, developed pseudoainhum which threatened the viability of her little fingers. This responded to treatment with etretinate. The treatment dilemma posed by keratoderma-induced pseudoainhum in children, i.e. the concern over the possible skeletal toxic effects of long-term etretinate treatment vs. the risks and outcome of surgery, is discussed.

Neutral lipid storage disease with ichthyosis: serum apolipoprotein levels and cholesterol metabolism in monocyte-derived macrophages.

Neutral lipid storage disease with ichthyosis (NLSDI) is an inherited metabolic disorder characterized by accumulation of neutral lipids, in a wide variety of cells, by a still unknown mechanism. Previous studies have shown normal cholesterol content in NLSDI granulocytes, fibroblasts and skin cells. Monocyte-derived macrophages possess an additional pathway of cholesterol uptake, which is not shared by these cells and which is not regulated by intracellular cholesterol levels. This pathway is thought to play a rôle in the process of atherosclerosis. Three NLSDI patients were studied. The serum levels of triglycerides, cholesterol, high-density lipoprotein cholesterol, and apolipoproteins A-I and B were within normal limits in all three patients. The intracellular levels of free and esterified cholesterol were measured in the monocyte-derived macrophages of one patient and found to be normal, while the triglyceride concentrations were twice as high as normal. The cholesterol esterification rates, which serve as a sensitive indicator of intracellular changes in cholesteryl ester levels, were normal in the monocyte-derived macrophages of all three patients. These findings provide further evidence that cholesterol metabolism is not disturbed in NLSDI, and it may be inferred that in this respect these patients are not at increased risk for atherosclerosis.

[Brown spider bite].

The diagnosis of bite by the brown recluse spider, Loxosceles reclusus, is rarely based on absolute identification of the insect because the victims are usually bitten while sleeping or dressing. More often, the history, clinical findings and course of the bite lead to the diagnosis. For early confirmation up to 24 hours after the bite, the passive hemagglutination test can be used. For older lesions, the in-vitro lymphocyte transformation test is useful, but is available in only a few medical centers. Treatment of the bite of the brown recluse spider varies from conservative to more active approaches. Resting, local cooling, systemic antibiotics to prevent infection and mild anti-inflammatory drugs may be given. In the more active approach oral corticosteroids are added in the first 72 hours to the antibiotics, especially in massive bites with necrotic centers greater than 2 cm in diameter, or when there is systemic loxoscelism. Recently, good results have been reported with Avlosulfon (dapsone), which is claimed to cure necrotic cutaneous ulcerations, presumably by reducing the activity of polymorphonuclear leukocytes. Other treatments include specific antivenin, (of limited use because it must be administered shortly after the bite), and surgery to prevent spreading of the venom. We describe 3 cases of brown spider bite with typical clinical presentations in adults aged 20-40 years. 2 were treated with corticosteroids and antibiotics and 1 with Avlosulfon and prednisone, all within 72 hours of the bite. 2 recovered completely within a few days, but the third treated with prednisone and antibiotics, developed an ulcer which healed only after several months of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune response to weakly immunogenic virally induced tumors. IX. Mice injected with the in vitro variant of YAC tumor (YAC-1) resist lethal doses of the tumorigenic YAC cells.

The ability of the in vitro propagated syngeneic tumor YAC-1 and the allogeneic tumor RBL5 to stimulate cytotoxic cells in A mice, was in correlation with the ability of these tumors to impose in vivo resistance to the viable YAC cells. Similarly, the ability of allogeneic YAC-1 tumor and syngeneic RBL5 tumor to stimulate cytotoxic cells in C57BL/6 mice was in correlation with the ability of these tumors to impose in vivo resistance to the viable RBL5 cells. In contrast, the original in vivo carried YAC tumor cells that induce the appearance of suppressor cells, as measured by in vitro assay, stimulated the in vivo enhancement of tumor growth in A mice. Adult thymectomy delayed the death of A mice injected with viable YAC cells, suggesting that at least some of the cells participating in the tumor enhancement are thymus derived cells.

Immune responses to weakly immunogenic murine-leukemia-virus-induced tumors. VII. Kinetic studies on various parameters of effects induced with suppressor cells.

YAC, a Moloney-virus-induced tumor of A mice, caused an inhibition of specific immunologic responses in A and C57BL/6 mice, which was mediated by suppressor cells. In contrast, YAC-1, the in vitro-carried tumor derived from cultivated YAC cells, stimulated the appearance of antitumor reactive cells in A mice. Splenocytes from YAC-1-injected mice generated anti-YAC cytotoxic cells after six days of culture. The suppressor cells from YAC-injected mice efficiently inhibited the cytotoxic responses of the cultivated reactive cells (YAC-1-primed cells) when added at the start of the culture, but not when added at later times. Suppressor cells appeared in A mice three days after injecion of YAC cells and persisted in the animals for at least 50 days. YAC-1-primed cells, derived from A mice 1, 3, 9 and 20 days after injection of YAC-1 cells, were sensitive to the suppressor cells.

Immune responses to weakly immunogenic murine-leukemia-virus-induced tumors. VIII. Characterization of suppressor cells.

YAC is a Moloney-virus-induced tumor of A mice. YAC-injected A and C57BL/6 mice generated suppressor cells. The suppressive function of the cells was tested by determining the ability of splenocytes from YAC-injected mice to inhibit the in vitro cytotoxic responses of primed splenocytes. It was found that suppressor cells passed through a nylon-wool column did not adhere to the plastic surface and resisted treatment with rabbit antimouse brain serum and guinea-pig complement. Therefore, the suppressor cells were defined operationally as "null" complement. Therefore, the suppressor cells did not demonstrate high efficiency; a relatively high concentration of suppressor cells was required to achieve an effective inhibiting action. These findings are discussed in terms of the heterogeneity of suppressor cells.

A feasibility study of prompt capture gamma in vivo neutron activation analysis.

The feasibility of using the information contained in the radiative capture gamma ray spectrum of the neutron-irradiated human body to measure quantitatively total body elemental composition in vivo has been investigated. Results of time dependent Monte Carlo simulations have shown that spectral interference of nonradiative capture origin can be completely eliminated by pulsing the detector/spectrometer system in anticoincidence with the neutron source. Calculations based on the results of the Monte Carlo simulation and on an experimental measurement of the efficiency versus energy characteristics of a Ge(Li) detector suggest that the primary limitation of the proposed technique would be inter-element spectral interference rather than inadequate detector sensitivity. Experimental measurements using a pulsed 14-MeV neutron generator and Ge(Li) gamma-ray spectrometer have produced results that are consistent with the predictions of the theoretical model. A radiative capture gamma-ray spectrum of a tissue-equivalent phantom was measured in pulsed mode and analyzed offline using a computerized spectral analysis procedure. The results were scaled to a proposed facility consisting of a 2.5-MeV pulsed neutron source and a detection system comprising two 50-cm3 (Ge(Li) detectros past which the subject would be scanned. It has been shown that in principle the elements hydrogen, chlorine, calcium, and nitrogen [the latter using large NaI(T1) detectors] could be measured with such a facility at an average body dose level of 0.1 rad. At this dose level the coefficients of variation based on counting statistics alone would be +/- 2%, or better, for these four elements. With an improvement in the detector/spectrometer energy resolution, the elements sulfur and carbon might also be measurable. It is also shown that by modifying the pulsing sequence appropriately and using 14-MeV neutrons, total body oxygen could also be measured at the 0.1 rad dose level via its inelastic neutron scattering deexcitation gamma activity.

Immune responses to weakly immunogenic virally induced tumors. II. Suppressive effects of the in vivo carried tumor YAC.

Unprimed spleen cells from A and C57BL/6 mice could not produce cytotoxic responses to their syngeneic tumors: a Moloney virus-induced in vitro subline YAC-1 and a Rauscher virus-induced in vitro subline RBL5, respectively. Spleen cells from A and C57BL/6 mice immunized with YAC-1 OR RBL5 (which cross-react serologically) generated significant syngeneic cytotoxicities after cultivation in vitro. The in vivo carried tumor of A mice, unlike the in vitro sublines, could not stimulate a priming effect. In contrast, YAC stimulated the formation of suppressor cells in both A and C57BL/6 mice. The suppressor cells abrogated the priming effect of the syngeneic tumors, but not the priming effect of the allogeneic tumors. Furthermore, YAC did not suppress normal allogeneic anti-tumor responses. The theoretical and the practical implications of these studies are discussed.