Endoscopic Islet Autotransplantation Into Gastric Submucosa-1000-Day Follow-up of Patients.

BACKGROUND: Total pancreatectomy and autologous transplantation of pancreatic islets is a treatment option for patients with severe pain due to chronic pancreatitis. In the standard procedure, pancreatic islets are isolated and subsequently administered into the portal vein. In the case of patients with a history of thrombosis or at risk of thrombosis, this route of administration is not viable. Animal studies conducted in our department led to the development of a technique of endoscopic islets transplantation into the gastric submucosa. In 2013 and 2014, the first human autologous transplant procedures were performed. The objective of this study was to present the results of a 3-year follow-up of these patients. METHODS: Two pancreatectomies were performed in our department, the first in 2013 and another in 2014, along with subsequent autologous transplantation of pancreatic islets into the gastric submucosa. RESULTS: Both patients had been diagnosed previously with diabetes, and both had endogenous islet activity detected. Peptide C concentration after pancreatectomy and before pancreatic cell transplantation was 0.1 ng/mL. After the transplantation, peptide C concentrations for the 2 patients were 0.8 and 0.5 ng/mL on day 7, 1.2 and 0.6 ng/mL on day 30, 1.3 and 0.8 ng/mL on day 180, 1.1 and 0.7 ng/mL on day 360, and 3.0 and 0.6 ng/mL at 3 years, respectively, after transplantation. The pain symptoms resolved in both cases. CONCLUSION: Pancreatic islets may survive in the gastric wall. Endoscopic submucosal transplantation may present an alternative for the management of patients who cannot undergo a classic transplantation procedure.

Islets Allotransplantation Into Gastric Submucosa in a Patient with Portal Hypertension: 4-year Follow-up.

BACKGROUND: Islets transplantation is an established treatment method for patients suffering from brittle diabetes with hypoglycemia unawareness. The standard implantation technique is through the portal vein into the liver. In case of liver diseases or portal hypertension, finding an extra-hepatic site is recommended. There have been attempts to perform islets transplantations into muscles and into the gastric submucosa. OBJECTIVE: The aim of this study is to show a 4-year follow-up of allotransplantation into gastric submucosa in a case of portal hypertension observed during the procedure of islets infusion. PATIENTS AND METHODS: A 36-year-old woman with complicated diabetes for over 30 years was selected to receive simultaneous islets and kidney transplantation. The patient underwent an unsuccessful simultaneous pancreas and kidney transplantation 2 years earlier in another transplantation center. The patient's daily insulin requirement was 60 IU, which corresponded to 1.15 IU/kg of body weight. The HbA1c level was 7.4%. C-peptide levels, both fasting and stimulated, were 0.01 ng/mL. On December 7, 2013, the patient received transplanted kidney and islets procured from the same donor. Only 124,000 islets equivalents (IEQ) were isolated (2400 IEQ/kg body weight). Islets were suspended in 300 mL of Ringer's solution along with albumin, antibiotics, and heparin. After infusing 100 mL of the islets suspension into the portal vein, pressure in portal vein increased from 5 mm Hg to 23 mm Hg. Despite stopping the infusion, pressure did not drop after 30 minutes. The decision was made to transplant the reminder of the islets (200 mL) into the gastric wall. RESULTS: No complications were observed after the procedure. Serum creatinine level was 1.6 mg/dL on day 10 and 1.5 mg/dL 4 years after the transplantation. Fasting C-peptide levels were 1.7, 0.65, 0.55, 0.69, 0.68, and 0.2 ng/mL at 1, 3, 6, 12, 18, and 36 months after the transplantation, respectively. HbA1c levels were 5.2, 6.4, 4.7, 5.2, and 5.9% at 3, 6, 12, 18, and 36 months, respectively. The patient's insulin requirement dropped to 15 U/day immediately after transplantation and equaled 20 and 27 U/day at 18 and 48 months after the simultaneous islet and kidney transplantation, respectively. CONCLUSION: Allotransplantation of islets into the gastric wall may be a safe alternative in cases of contraindications for transplantation into the portal vein.

Replicating reoviruses with a transgene replacing the codons for the head domain of the viral spike.

The capacity to modify the reovirus genome facilitates generation of new therapeutic reoviruses. We describe a method for generating replication-competent reoviruses carrying a heterologous transgene. The strategy is based on the expanded-tropism reovirus mutant jin-3, which can infect cells independent of the reovirus receptor junction-adhesion molecule A (JAM-A). Jin-3 harbors a mutation in the S1 segment, resulting in a G196R substitution in the tail of the spike protein σ1. The use of the jin-3 tail-encoding S1 segment allows replacing the codons for the JAM-A-binding head domain by up to 522 nucleotides of foreign sequences, without exceeding the size of the wild-type S1 segment. We inserted the codons for the porcine teschovirus-1 2A element fused with those encoding the fluorescent protein iLOV. Replicating rS1His-2A-iLOV reoviruses were generated by co-transfection of expression plasmids for all reovirus segments. These reoviruses contain the S1His-2A-iLOV segment in the absence of the wild-type S1 segment. Density-gradient centrifugation confirmed the association of the σ1-tail fragment with the capsid. Both JAM-A-positive and -negative cells exposed to the rS1His-2A-iLOV reoviruses exhibited iLOV fluorescence, confirming the jin-3-derived expanded-tropism phenotype. These data demonstrated the feasibility of generating decapitated replication-competent T3D reoviruses carrying a heterologous transgene.

Pro- and antiangiogenic markers in patients with pulmonary complications of systemic scleroderma.

Systemic sclerosis (SSc) is an autoimmune disorder characterized by skin and internal organs fibrosis and concomitant vascular abnormalities. Although SSc is considered mainly fibrosing disease, underlying vascular pathology plays a fundamental role in its pathogenesis. We have focused on positive and negative serum markers of angiogenesis and fibrosis (pigment epithelium-derived factor [PEDF], vascular endothelial growth factor [VEGF], and soluble VEGF receptor [sVEGFR]), in progressive SSc patients at baseline and after follow-up in relation to cardiopulmonary complications (systemic hypertension [HT], pulmonary arterial hypertension [PAH] and pulmonary fibrosis [PF]). VEGF and PEDF but not sVEGFR were reciprocally regulated in SSc progression. Moreover, VEGF/PEDF ratio significantly increased during follow up suggesting that it might be used as a biomarker of disease progression. No correlation between the studied markers and cardiopulmonary complications was observed. In conclusion, VEGF and PEDF level, and the VEGF/PEDF ratio are significantly changed in the course of SSc progression and these markers can be used to assess SSc activity.

3-Fluoromethcathinone, a structural analog of mephedrone, inhibits growth and induces cell cycle arrest in HT22 mouse hippocampal cells.

Recently, the abuse of recreational drugs has become an important problem in many countries. Among these psychoactive substances are synthetic cathinones, a group of compounds derived from the alkaloid cathinone, that have gained widespread popularity. Many cathinones have demonstrated neurotoxic effects. The aim of this study was to examine the effects of 3-fluoromethcathinone, a structural analog of mephedrone, on HT22 mouse hippocampal cells. Cell viability was assessed using the sulforhodamine B assay. Flow cytometry was used to study the cell cycle distribution. We found that 3-fluoromethcathinone inhibits growth of HT22 cells. Our results also revealed that it induces G0/G1-phase cell cycle arrest. To our knowledge, this is the first study to demonstrate the cytotoxic action of 3-fluoromethcathinone. Our findings suggest that abuse of this cathinone derivative may not be without risk.

Kinetics of calcium ion concentration accompanying transduction of signals into neutrophils from diabetic patients and its modification by insulin.

The goal of the study was to evaluate the process of Ca(2+)-mediated transduction of signals into neutrophils from patients with type I diabetes and its modification by insulin. The study was performed with the use of isolated peripheral blood neutrophils from 20 diabetic patients and 30 healthy volunteers. Isolated granulocytes were stimulated separately by fMLP or insulin, or by both substances added to the medium in combinations: fMLP + insulin (after 20 min) or insulin + fMLP (after 20 min). fMLP evoked fast intracellular increase of free Ca(2+) concentration in neutrophils compared with the resting state (P<0.001). Similarly, the peak of fluorescence, as measured by Fluo 3 to Fura Red ratio, was significantly higher in neutrophils stimulated by insulin. Insulin did not cause any changes in intracellular Ca(2+) level when it was added to the previously fMLP-stimulated cells. Prestimulation with insulin significantly decreased fMLP-induced intracellular free Ca(2+) concentration, expressed as Fluo3/Fura Red ratio compared with fMLP alone (1.77 +/- 0.6 vs. 2.63 +/- 0.8, P<0.001). No relation between initial intracellular Ca(2+) in the resting state and the response to insulin was found. Nor was the response to fMLP alone related to intracellular Ca(2+) before stimulation. A strong correlation was observed between initial intracellular Ca(2+) after incubation with insulin and the response to fMLP (r=0.90, P<0.0001). In diabetic granulocytes, the intracellular Ca(2+) was significantly lower than in those from healthy donors in unstimulated cells (P<0.001), after fMLP stimulation (P<0.0001), in medium enriched by insulin (P<0.05), and after fMLP stimulation in insulin rich medium (P<0.001). Only in fMLP prestimulated samples, the emission of light did not differ after stimulation with insulin in granulocytes from both diabetic and healthy subjects. In conclusion, patients with type I diabetes have decreased levels of cytosolic Ca(2+) after insulin and fMLP stimulation in polymorphonuclear granulocytes. This abnormality is probably primarily responsible for the impaired neutrophilic function seen in these patients.

The influence of insulin on calcium ion concentration during transduction of signals into neutrophils.

The goal of the study was to evaluate the process of neutrophil activation via Ca(2+)-mediated transduction signal and its modification by insulin. The study was performed with the use of isolated peripheral blood neutrophils obtained from 20 healthy volunteers. Isolated granulocytes were stimulated by fMLP or insulin alone, or by both substances added to the medium in combinations: fMLP + insulin (after 20 min) or insulin + fMLP (after 20 min). To explore the mechanism of intracellular Ca(2+) changes, receptor signal transduction pathway was blocked by tyrosine kinase inhibitors: tyrphostin 25 and genistein. fMLP evoked fast intracellular increase of free Ca(2+) concentration in neutrophils, compared with the resting state (P< 0.001). Insulin did not cause any changes in intracellular Ca(2+) when was added to the previously fMLP stimulated cells. Prestimulation with insulin significantly decreased fMLP-induced intracellular free Ca(2+) concentration compared with fMLP alone (P<0.01). A strong correlation was observed between initial intracellular Ca(2+) concentration after incubation with insulin and the response to fMLP (P<0.0001). The tyrphostin 25 did not influence the Ca(2+) concentration in control granulocytes, but inhibited the fMLP-induced intracellular Ca(2+) increase when added before fMLP (P<0.05). In a Ca(2+)-free medium, a strong relationship between intracellular Ca(2+) and the response to fMLP after incubation with tyrphostin was found (P<0.001) The genistein did not influence the intracellular Ca(2+) in non-stimulated cells. However, it inhibited the fMLP-induced Ca(2+) increase when added before fMLP (P<0.05). The genistein added to the suspension of cells after fMLP stimulation did not influence intracellular Ca(2+) level. A positive correlation was found between the initial intracellular Ca(2+) and the response to fMLP of genistein preincubated cells. This effect was seen in both Ca(2+)-rich, and Ca(2+)-free medium We conclude that insulin is a potent immunomodulator and its signaling pathways are mediated by Ca(2+) concentration changes. The process of intracellular Ca(2+) changes following insulin signaling is, at least partly, tyrosine kinase-related. Derangements in the concentration of intracellular Ca(2+) may represent a link between the mechanisms of insulin resistance in diabetes.

Differences in glycosylation of intracellular proteins between benign and malignant thyroid neoplasms.

Differences in glycosylation of nuclear and cytosolic proteins isolated from benign and malignant human thyroid neoplasms were analyzed by lectin blotting and enzyme linked lectino-solid-phase assay using Erythrina cristagalli and Ricinus communis agglutinins. The results reported in this study have not shown any significant differences in lectin binding by nuclear proteins of benign and malignant tumors, however, quantitative and qualitative differences were observed in the patterns of cytosolic glycoproteins. In the majority of carcinomas samples lectin binding to cytosolic proteins was definitely weaker in comparison with adenomas and non-neoplastic specimens, which suggested alterations in glycosylation of cytosolic proteins in malignant tumors.

Glycoproteins of chicken liver nuclei cross-linked to DNA by cis-diamminedichloroplatinum.

Glycoproteins which participate in DNA-protein cross-links induced by action of cis-diamminedichloroplatinum (cis-DDP) in intact nuclei of chicken liver were investigated. Digoxigenin-labelled lectins with different sugar specificity were used for detection and characterization of these glycoproteins. Our results showed the presence of glycoproteins bearing high mannose as well as complex type oligosaccharides in chicken liver nuclei. In most cases of complex oligosaccharides, sialic acid residues bound in alpha(2-6) but not in alpha(2-3) linkage were present.

Glycoproteins present in the fraction of chromatin proteins loosely bound to DNA from hamster, chicken and frog liver cells.

There are numerous glycoproteins recognized by Concanavalin A (ConA) and Galanthus nivalis agglutinin (GNA) in 0.35 mol/l NaCl soluble fraction of chromatin proteins loosely bound to DNA from hamster, chicken and frog liver cells. Results of our detailed comparative analysis show a marked similarity between liver chromatin glycoproteins from the examined animals. The presence of similar chromatin glycoproteins in different animal species may indicate that they play an important universal role in the liver cells.

Nuclear liver glycoproteins at different stages of chicken embryo development.

In order to examine whether the patterns of nuclear and chromatin glycoproteins change during development the glycoproteins of foetal and adult chicken liver were investigated. Nuclear and chromatin proteins from both sources were separated by SDS-PAGE, transferred onto Immobilon-P transfer membrane or nitrocellulose and tested for concanavalin A (Con A), Galanthus nivalis agglutinin (GNA) and Aleuria aurantia agglutinin (AAA) binding. Results revealed a similarity in the profiles of nuclear and chromatin glycoproteins recognized by Con A from 14-, 16-, 18-day foetal and adult chicken liver. Generally GNA and AAA reacted more weakly with glycoproteins from foetal liver compared with the same glycoproteins from adult liver.

[Microalbuminuria as a risk factor for diabetic osteopathy in patients with IDDM and renal sufficiency]. / Mikroalbuminuria jako czynnik ryzyka osteopatii cukrzycowej u pacjentów z IDDM w okresie wydolnosci nerek.

Disturbances in bone marrow vascularisation can be one of the causes of diabetic osteopathy. The aim of the study was to answers the question if microalbuminuria as a results of capillary injury can be a sign of bone mineralisation disorders in IDDM renal sufficient patients. We examined 60 IDDM patients (30 women without menstruation disturbances; 30 men; age 25-36 years old). All the observed subjects were divided into groups: I-30 normoalbuminuric patients (0-29 mg/24 h); II-30 microabuminuric patients (30-295 mg/24 h). Bone mineral density (BMD) of femoral neck, lumbar spine (L2-L4) and total body was measured by dual energy X-ray absorptiometry (DEXA, Lunar). The biochemical parameters of bone turnover were measured both in serum and urine as follows: osteocalcine, total hydroxyproline (HPR, HPR/Cr), total alkaline phosphatase (AP) with bone fraction, total calcium (Ca, Ca/Cr) and inorganic phosphor (P). Microalbuminuric patients presented more severe bone turnover disturbances, shown by differences in: BMD and Z-score for femoral neck (p < 0.05), serum HPR (p < 0.05), AP (p < 0.05), AP (p < 0.01) and its bone fraction (p < 0.05). We proved the presence of statistically significant correlation coefficients for albuminuria and some densytometric and biochemical bone parameeters. Our results suggest that microalbuminuria can indirectly indicate the dynamic of bone turnover derangement in IDDM course. They are present mostly in the femoral neck, which because of the vascularisation type is particularly susceptible to subalimentation in the diabetic microangiopathy course.

[Bone mineralization in insulin-dependent diabetes mellitus]. / Mineralizacja tkanki kostnej w cukrzycy insulinozaleznej.

One of insulin-dependent diabetes mellitus (IDDM) complications are bone mineral disorders called diabetic osteopathy. Because of many controversies of this subject we took a research of bone mineralisation in IDDM patients before 40 years old. The evaluation of BMD was performed with the use of X-ray dual energy absorptiometry--DEXA in the AP projection for lumbar part of vertebral column (BMD L2-L4), left femur neck (BMD-neck) and total skeleton (BMD-total). Bone tissue metabolism was evaluated with the aid of biochemical tests. The examined group consisted of 99 patients with IDDM (45 women and 54 men), without any other risk factors for changes in bone metabolism. The results were related to: sex, age in which IDDM was diagnosed, duration of IDDM, metabolic control of diabetes and to some IDDM complications. The control group consisted of 113 healthy subjects matched for age, sex, weight, height and calcium diet. We observed that BMD in IDDM patients before 40 years old was significantly lower than in healthy subject. One of some diabetic osteopathy pathomechanism seems to be an advantage of bone resorption over bone formation. Bone demineralisation which was observed to be most pronounced in femur neck, was not related to age, differed in accordance to sex and was higher of age when IDDM appeared was lower. BMD was also related to duration of IDDM and metabolic control of IDDM.

Effect of carbohydrate moieties on the stability of nuclear glycoproteins from hamster liver.

The effect of carbohydrate moieties on the stability of hamster liver nuclear glycoproteins in the course of endogenous proteolysis employing highly specific digoxigenin-labelled lectins, was studied. Whole hamster liver nuclei were autolysed in optimum conditions for the action of nuclear proteinases able to degrade histones as well as non-histone proteins. Incubated samples were electrophoresed on sodium dodecyl sulphate-polyacrylamide gel. Coomassie blue stained gels demonstrated degradation of some proteins in particular after 18 and 24 h incubation. Proteins with molecular weights of about 46, 54 and 76 kD appeared to be resistant to proteolysis. The same location and intensity of bands of glycoproteins on immunoblots from incubated and nonincubated samples of nuclei indicated that oligosaccharide chains protect proteins from degradation.

Age-related patterns of lectin-binding nuclear glycoproteins of hamster liver.

We studied the age-related patterns of lectin-binding liver nuclear glycoproteins from hamsters between 5 and 35 weeks of age. The examinations of the carbohydrate structures of liver nuclear glycoproteins in relation to the age of hamsters were carried out after electrophoresis and blotting by a very sensitive immunological detection system with highly specific digoxigenin-labelled lectins. The results reported in the present study do not show any significant changes in the patterns of nuclear glycoproteins with regards to the age of hamsters except the decrease in affinity of wheat germ agglutinin (WGA) to 63 kDa glycoprotein bearing single O-linked N-acetylglucosamine residues.

[Sneddon syndrome]. / Zespóll sneddona.

There is presented a case a 42 year old woman, who was admitted to the Department of Internal Diseases, the Institute of Dentistry, Medical Academy in Warsaw, with suspected bacterial endocarditis. Two episodes which indicate a lesion in the central nervous system (as right side hemiparesis and mixed aphasia) in the patients with valvular heart disease, were the basis of this suspicion. Bacterial endocarditis was not confirmed during hospitalisation. Diagnosis of Sneddon's syndrome was established based on skin lesion of livedo reticulatis type with typical picture in skin biopsy and on the lesions in the central nervous system confirmed by MRI. Moreover the patients had arterial hypertension and Raynaud's syndrome. We presented the diagnostic difficulties of Sneddon's syndrome, a course of the disease, the factors, which affect its prognosis and attempts to treat this syndrome based on this case.

Lectin-binding glycoproteins in nuclear fractions from hamster liver and Kirkman-Robbins hepatoma.

As a further step toward characterizing the major nuclear glycoproteins from hamster liver and Kirkman-Robbins hepatoma (Lipinska A. and Gaczynski M. Int. J. Biochem. 4, 1385-1390, 1992) its intranuclear localization was studied. The glycoprotein patterns of examined nuclear fractions of hamster liver and hepatoma revealed some cell specificity observed especially in nuclear matrix preparations. Our results show the extensive presence of envelope glycoproteins in the nuclear matrix.

Nuclear glycoproteins of hamster liver and Kirkman-Robbins hepatoma recognized by concanavalin A.

1. Glycoproteins recognized by Concanavalin A (ConA) have been identified in nuclei and nuclear fractions differing in sensitivity to micrococcal nuclease digestion from hamster liver and Kirkman-Robbins hepatoma. 2. The major ConA binding proteins from hamster liver and Kirkman-Robbins hepatoma nuclei have molecular weights about 27,000 and 57,000, and 38,000 and 49,000, respectively. 3. A distinct distribution of glycoproteins between fractions differing in sensitivity to nuclease digestion has not been observed.

Nuclear distribution pattern of tumour-associated nonhistone protein of mol. wt 48,000.

1. As a further step toward characterizing nonhistone protein of mol. wt 48,000 which was found to be much more abundant in animal tumour cells than in normal ones [Krajewska W.M., Lipínska A., Marszatek M., Kilianska Z., Wojtkowiak Z. and Klyszejko-Stefanowicz L. Cell. Biochem. Funct. 8, 79-89 (1990)] its intranuclear localization in hamster liver and Kirkman-Robbins hepatoma was studied. The protein was identified by immunoblotting technique in the presence of antibodies against polypeptide of mol. wt about 48,000 from Kirkman-Robbins hepatoma. 2. Distribution of antigen with mol. wt of 48,000 in nuclear fractions representing different levels of nuclear material organization, i.e. in nucleoli, nuclease-sensitive and nuclease-resistant fractions, and extensive nuclease digestion products separated by size on Bio-Gel A-50m; implied the structural role of this component. 3. Fractionation of endogenously digested nuclei into low salt extract, high salt extract and nuclear matrix revealed that in normal liver the antigen studied is associated with nuclear matrix while in hepatoma this component appeared in high salt extract. 4. These results suggest that polypeptide with mol. wt of 48,000 is a shuttling protein which may be involved in reorganization of nuclear matrix during neoplastic transformation.

Nuclear proteins of hamster hepatoma after administration of antitumour agents.

One- and two-dimensional gel electrophoretic analysis of nuclear proteins of Kirkman-Robbins hepatoma was used to study the effects of the tumour growth inhibitors methotrexate (MTX) and acyclonucleoside (DHPQtB) on protein composition. MTX and DHPQtB inhibited Kirkman-Robbins hepatoma growth by 89.2 +/- 3.5% and 16.3 +/- 6.1% respectively. The biosynthesis and/or metabolism of some polypeptide spots was affected by these antitumour agents, especially among components with molecular wt/isoelectric points of 52,000-64,000/4.9-5.5, 69,000-78,000/5.0-5.9 and 88,000-100,000/5.1-5.9.