Using methylcholanthrene-induced fibrosarcomas to study tumor immunology.

Mouse models of cancer are essential in furthering our understanding both of the mechanisms that drive tumor development and the immune response that develops in parallel, and also in providing a platform for testing novel anti-cancer therapies. The majority of solid tumor models available rely on the injection of existing cancer cell lines into naïve hosts which, while providing quick and reproducible model systems, typically lack the development of a tumor microenvironment that recapitulates those seen in human cancers. Administration of the carcinogen 3-methylcholanthrene (MCA), allows tumors to develop in situ, forming a tumor microenvironment with an established stroma and vasculature. This article provides a detailed set of protocols for the administration of MCA into mice and the subsequent monitoring of tumors. Protocols are also provided for some of the routinely used downstream applications that can be used for MCA tumors.

Development of ultrasound enthesitis score to identify patients with enthesitis having spondyloarthritis: prospective, double-blinded, controlled study.

OBJECTIVES: To distinguish patients (pts) with enthesitis having spondyloarthritis (SpA) from pts with enthesitis without SpA by ultrasound (US) enthesitis score. METHODS: The study sample included 127 pts with enthesitis (76 pts with SpA, 26 pts with rheumatoid arthritis, 25 pts with mechanically-related enthesitis). The entheses of plantar fascia, Achilles, patellar, quadriceps and common extensor tendon on lateral epicondyle were examined by US. Two operators, blinded to clinical diagnosis and enthesitis symptoms, assessed enthesis thickness, echogenicity, enthesophytes, power Doppler signal and erosions. Logistic regression and receiver operating characteristic (ROC) curve analysis were used to determine the predictive value of each enthesitis lesion for diagnosis of SpA. The best predictive value for SpA was accomplished when absence and presence of increased thickness, hypoechogenicity and enthesophytes were scored as 0 and 1; absence and presence of PD and erosions were scored as 0 and 4. Belgrade Ultrasound Enthesitis Score (BUSES) represents a cumulative score of derived enthesitis lesion scores at examined entheses. Independent-samples t-test was used for BUSES comparison between pts with and without SpA. Validity of BUSES for SpA diagnosis was evaluated by sensitivity and specificity. Cut-off point was chosen as the smallest value with specificity of at least 90%. The reliability was analysed by intra-class-correlation coefficient (ICC). RESULTS: BUSES was 9.9 ± 12.4 (mean ± SD) in SpA pts and 3.1 ± 4.2 in pts without SpA (p<0.001). BUSES cut-off point ≥ 7 achieved excellent specificity (90.2%) and fair sensitivity (47.4%). ICC was 0.99. CONCLUSIONS: BUSES is highly specific, valid and reliable to identify patients with SpA.

Dietary factors and gastric cancer risk: hospital-based case control study.

PURPOSE: To examine possible associations between dietary factors and gastric cancer risk among residents in the area of Nis, Serbia. METHODS: This hospital based case-control study was conducted at the Clinical Centre Nis between 2005 and 2006. Cases (n=102) with histologically confirmed gastric cancer and matched non-cancer patients (controls, n=204) were interviewed. Diet was assessed using a food frequency questionnaire and odds ratios (OR), 95% confidence interval (95% CI), and p-trends were calculated across tertiles of intake. RESULTS: High intake of salt, salty meals and hot food were associated with higher risk of gastric cancer. After searching for potential confounders, multivariate logistic regression analysis showed elevated risk for the highest vs. the lowest tertile of intake for smoked and barbecue meat (OR 4.21; 95% CI 1.43-12.37), processed meat (OR 9.17; 95% CI 2.78-15.23), desert (OR 2.85; 95% CI 1.28-6.38), potatoes (OR 4.79; 95% CI 1.44-5.94), pickled vegetables (OR 2.02; 95% CI 1.21-3.0) and milk (OR 5.08; 95% CI 1.59-10.16) intake but reduced for citrus fruits (OR= 0.13, 95% CI 0.03- 0.53), other fruits (OR 0.05; 95% CI 0.02-0.18), allium vegetables (e.g. onion, garlic, leek) (OR 0.11; 95% CI 0.02-0.60) and cooked meat intake (OR 0.07; 95% CI 0.02-0.27). Intake of bread, dairy, fish, legumes and raw and cooked vegetables was not significantly related with gastric cancer risk. CONCLUSION: A low risk diet for gastric cancer in the area of Nis should include increased fruits and alliums vegetables consumption and limited processed meat, salt, preserved food, deserts, potatoes and milk intake.

Reliability of ultrasonography in detecting shoulder disease in patients with rheumatoid arthritis.

OBJECTIVE: To assess the intra and interobserver reproducibility of musculoskeletal ultrasonography (US) among rheumatologists in detecting destructive and inflammatory shoulder abnormalities in patients with rheumatoid arthritis (RA) and to determine the overall agreement between US and MRI. METHODS: A total of 14 observers examined 5 patients in 2 rounds independently and blindly of each other. US results were compared with MRI. Overall agreement of all findings, of positive findings on MRI, as well as intra and interobserver reliabilities, were calculated. RESULTS: Overall agreement between US and MRI was seen in 79% with regard to humeral head erosions (HHE), in 64% with regard to posterior recess synovitis (PRS), in 31% with regard to axillary recess synovitis (ARS), in 64% with regard to bursitis, in 50% with regard to biceps tenosynovitis (BT), and in 84% for complete cuff tear (CCT). Intraobserver and interobserver kappa was 0.69 and 0.43 for HHE, 0.29 and 0.49 for PRS, 0.57 and 1.00 for ARS, -0.17 and 0.51 for bursitis, 0.17 and 0.46 for BT and 0.52 and 0.6 for CCT, respectively. The intraobserver and interobserver kappa for power Doppler (PD) was 0.90 and 0.70 for glenohumeral signals and 0.60 and 0.51 for bursal signals, respectively. CONCLUSIONS: US is a reliable imaging technique for most shoulder pathology in RA especially with regard to PD. Standardisation of scanning technique and definitions of particular lesions may further enhance the reliability of US investigation of the shoulder.

Gatifloxacin 200 mg or 400 mg once daily is as effective as ciprofloxacin 500 mg twice daily for the treatment of patients with acute pyelonephritis or complicated urinary tract infections.

The efficacy and safety of two oral dosing regimens of gatifloxacin were compared to ciprofloxacin in the treatment of complicated urinary tract infection in a randomised, double-blind multi-centre trial. One thousand one hundred and twenty-three adult patients with complicated urinary tract infection (70%) or pyelonephritis (30%) were initially enrolled, 1122 were treated. Of these, 824 were included in a modified ITT population: gatifloxacin 200 mg (274 patients) or 400 mg (280 patients) once daily or ciprofloxacin 500 mg twice daily for 5-14 days (269 patients). Bacteriological and clinical responses were assessed 7-9 days after the end of treatment (EOT) and 4-6 weeks post-treatment (end of study visit, EOS). The bacteriological response rates per patient at EOT in the gatifloxacin 400 mg, gatifloxacin 200 mg and ciprofloxacin groups were 77% (207/269), 78% (208/268) and 73% (190/259), respectively. At EOS they were slightly lower: 70% (184/262), 71% (176/248) and 69% (174/252), respectively. The clinical responses at EOT were 69% (190/277), 70% (190/273) and 65% (174/266). At EOS they were 71% (193/273), 70% (182/259) and 74% (190/258). The overall eradication rates of initial pathogens at EOT and EOS were 85.3% and 88.4% in the gatifloxacin 400 mg group; 84.1 and 90.1% in the gatifloxacin 200 mg group and 85.1 and 91.4% in the ciprofloxacin group. Both oral regimens of gatifloxacin were as effective as that of ciprofloxacin. All treatment groups showed a similar safety profile, nausea being the most frequently reported adverse event.

[Primary CNS lymphomas. Morphology and diagnosis]. / Primäre ZNS-Lymphome. Morphologie und Diagnostik.

Diagnostically primary central nervous system lymphomas (PCNSL) have to be differentiated from glioblastoma and brain metastases. Histologically the overwhelming majority of PCNSL is represented by diffuse large B-cell lymphomas, in this series with a BCL6 expression in 80% of the cases detected by immunohistochemistry. Stereotactic biopsy is the method of choice in establishing the definite diagnosis and intraoperative smear cytology will detect the lymphoid blasts. To confirm the B-cell lineage, immunohistochemistry is needed (CD20, CD79a). Small reactive T-lymphocytes and monohistiocytic cells and activated "microglia" are found within and at the periphery of PCNSL foci. The infiltrated brain tissue shows partially pleomorphic reactive astrocytes that can be confused with neoplastic astrocytes, especially in small specimens. In contrast to high-grade gliomas, however, PCNSLs do not show endothelial proliferations. Subtypes or variants of diffuse large B-cell lymphomas can also be observed in cases of PCNSL: the anaplastic variant with large multinucleated tumour cells resembling Reed-Sternberg cells, T-cell rich B-cell lymphoma and intravascular B-cell lymphoma with primary manifestation within the brain or the spinal cord. HIV/AIDS-associated PCNSLs are characterised by large plasmoblastic or small Burkitt-like cells and tumour necrosis. Primary leptomeningeal large B-cell lymphomas do occur very rarely and are diagnosed by cerebrospinal fluid cytology.

The DNMT1 target recognition domain resides in the N terminus.

DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. The N-terminal domain has been postulated to have a regulatory role, and it has been suggested that the mammalian DNMT1 is a fusion of a prokaryotic methyltransferase and a mammalian DNA-binding protein. Using a combination of in vitro translation of different DNMT1 deletion mutant peptides and a solid-state hemimethylated substrate, we show that the target recognition domain of DNMT1 resides in the N terminus (amino acids 122-417) in proximity to the proliferating cell nuclear antigen binding site. Hemimethylated CGs were not recognized specifically by the postulated catalytic domain. We have previously shown that the hemimethylated substrates utilized here act as DNMT1 antagonists and inhibit DNA replication. Our results now indicate that the DNMT1-PCNA interaction can be disrupted by substrate binding to the DNMT1 N terminus. These results point toward new directions in our understanding of the structure-function of DNMT1.

How does DNA methyltransferase cause oncogenic transformation?

Global hypomethylation of genes and repetitive sequences, as well as hypermethylation of certain genes known to be involved in tumor suppression, are observed concurrently in cancer cells. Aberrant expression of DNA methyltransferase 1 (dnmt1) is a downstream effector of multiple tumorigenic pathways, and several data suggest that dnmt1 plays a causal role in these pathways. These data raise two critical questions: Why does ectopic expression of dnmt1 transform cells? and How can global hypomethylation exist in a cell that bears high levels of DNMT1 activity? It is proposed that DNMT1 induces cellular transformation by a mechanism that does not involve DNA methylation and that the low level of methylation in cancer cells is a result of induction of a DNA demethylase in these cells. Both DNMT1 and the demethylase play a causal role in cellular transformation and are candidate anticancer targets.

DNA methyltransferase inhibition induces the transcription of the tumor suppressor p21(WAF1/CIP1/sdi1).

Previous lines of evidence have shown that inhibition of DNA methyltransferase (MeTase) can arrest tumor cell growth; however, the mechanisms involved were not clear. In this manuscript we show that out of 16 known tumor suppressors and cell cycle regulators, the cyclin-dependent kinase inhibitor p21 is the only tumor suppressor induced in the human lung cancer cell line, A549, following inhibition of DNA MeTase by a novel DNA MeTase antagonist or antisense oligonucleotides. The rapid induction of p21 expression points to a mechanism that does not involve demethylation of p21 promoter. Consistent with this hypothesis, we show that part of the CpG island upstream of the endogenous p21 gene is unmethylated and that the expression of unmethylated p21 promoter luciferase reporter constructs is induced following inhibition of DNA MeTase. These results are consistent with the hypothesis that the level of DNA MeTase in a cell can control the expression of a nodal tumor suppressor by a mechanism that does not involve DNA methylation.

Features of uremic neuropathy in long-term dialysis.

In order to assess the influence of long term hemodialysis on progression of uraemic neuropathy (UN), 16 different electroneurographic (ENG) parameters on 158 dialysis patients were performed. The ENG parameters were compared in three groups of patients of different dialysis age. Group I: high dialysis age (HDA) comprising of, 31 patients being more than 10 years on dialysis; Group II: intermediate dialysis age (IDA) comprising of 53 patients between 5 and 10 years on HD and group III: low dialysis age (LDA) comprising of 74 patients being less than 5 years on dialysis. The influence of sex and age was also analyzed. All sixteen tested parameters were altered in uremic patients when compared to 140 healthy controls (p < 0.01). HDA pts compared to LDA pts and the older group versus the younger had 11 and 9 out of 16 ENG parameters significantly worsened, respectively (p < 0.01). The most profound and reproducible lesion was in prolongation of evoked potential of tibialis posterior and peroneus nerve (FWt, HWt, FWp). HDA, especially after 10 yrs and the age but not the sex is clearly associated with a further progression of UN. However, for unknown reasons, the progression of UN in dialyzed patients is not followed by a parallel worsening of clinical symptoms (p > 0.05).

Pharmacokinetic behaviour of R-(+)- and S-(-)-amlodipine after single enantiomer administration.

Amlodipine, 3-ethyl 5-methyl-2-[(2-aminoethoxymethyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methy l-3,5-pyridinedicarboxylate, is a chiral calcium antagonist, currently on the market and in therapeutic use as a racemate. The pharmacokinetic behaviour of R-(+)- and S-(-)-amlodipine after single enantiomer administration to healthy male human volunteers together with comparative administration of the racemic mixture of both enantiomers were studied. Plasma levels were studied as a function of time and assayed using an enantioselective chromatographic method (coupled chiral and achiral HPLC) with on-line solid-phase extraction and UV absorbance detection. The method was validated separately for the R-(+)- and S-(-)-enantiomer, respectively. Results of the study indicate that the pharmacokinetic behaviour of R-(+)- and S-(-)-amlodipine after single enantiomer administration is comparable to that of each enantiomer after administration of the racemate. No racemization occurs in vivo in human plasma after single enantiomer administration.

[Verrucous carcinoma of the larynx]. / Verukozni karcinom larinksa.

In spite of a small percentage of laryngeal cancer, verrucous carcinoma of the larynx is an important diagnostic and therapeutic issue, due to its specific biological behavior and reaction to different therapeutic procedures. Verrucous carcinoma is free of histologic characteristics of malignancy, the lymph node and visceral metastasis. In our series of laryngeal cancer, we had 2.47% of verrucous carcinoma. The glottis was involved in all cases, and the treatment was surgical. Histologic characteristics of the tumour are discussed, and our clinical material analysed. The importance of a precise diagnosis for the correct selection of the therapeutic approach, is stressed.

[The beginnings of otorhinolaryngology in Bosnia and Herzegovina and its development in Sarajevo]. / Zaceci otorinolaringologije u Bosni i Hercegovini i njen razvoj u Sarajevu.

The first ENT Department in Bosnia and Herzegovina was established in Sarajevo by Dr. Zarko Prastalo in 1923, as a part of the Surgery Clinic, with 10 beds. The next year (1924) ENT got its own operation theatre, 30 beds more and a new localization. In that period there was only one ENT specialist, Dr. Zarko Prastalo. Three years after some new doctors started the specialization in ENT. By the year 1946 the ENT Department became a University clinic with 7 doctors on duty, the Clinic became an educational centre. To the year 1992, 112 ENT specialists were educated in the ENT Clinic in Sarajevo. Later in time, after Dr. Zarko Prastalo, Dr. Ivo Herlinger, Dr. Josip Gerc and Dr. Emilija Bac became known ENT specialists. The new scientific and operation techniques came to the ENT Clinic, the first chelognapalato schisis was in 1936, done by Dr. Josip Gerc. The first ear operation using operation microscope and microinstruments was performed in 1962, while the first laryngo-micro-operation in 1971. Since 1965 th worked with the Maxilofacial Surgery, together, but in 1972 they moved to the stomatologic Clinic, With the development of the clinic, 10 our patient departments (ambulances) were established in Sarajevo. As for now, during the war, there are 101 beds, 13 ENT specialists. Dr. Sejo Sebic was killed in 1992 by the aggressor.

Oxalosis associated with aluminum bone disease: a new type of mixed renal osteodystrophy.

An 18-year-old man suffering from uremia was hospitalized because of severe skeletal pain and multiple exostoses. The patient had been treated for 5 years by regular hemodialysis; primary oxalosis was the suspected cause of uremia. Transiliac bone biopsy and subsequent histomorphometric analysis of undecalcified bone specimen as well as specific aluminum staining revealed a picture of oxalosis combined with osteomalacia and a marked accumulation of aluminum at the front of mineralization. This combination of histological features of oxalosis and aluminum bone disease suggests a new type of mixed renal osteodystrophy.

[Management of soft-tissue defects related to war injuries]. / Zbrinjavanje mekotkivnih defekata ratnih povreda.

The paper analyzed our experiences with repairing the skin defects in 120 wounded patients in the first war year. After primarily surgical observations patients were hospitalized in the ORL clinic for a further care. Most of wounds were by artillery projectiles. Extensivity, intensivity and primary contamination are three characteristics of war wounds that makes problems in definitely repairing of defects. This is because of the secondary repair 67.80% defects. Spontaneous reepithelization is noticed in 14.80% defects. By direct sutures we closed 16.00%, free skin tissues grafts 23.26%, used various techniques of rotation and transposition flaps 46.52% defects. Correct plastic reconstructive planning and active physical-chemical preoperative treatment eliminated big postoperative complications.

[Care of war injuries of the nasal apparatus]. / Zbrinjavanje ratnih povreda nosnog kompleska.

Prominent position of nose and paranasal sinuses (nose complex) is one of the most percentage of war injuries (37.00% of ORL region). Unfortunately, these wounds are badly observed. So late we have big functional and aesthetical disorders. Using simple methods like inspection, palpation, rhinoscopy and some radiologic examination we could have a good information about kind and lesion extension. It is very important in operating planning. This paper is retrospective analyses of nose complex war injuries in a first 12 months of aggression. They are open nonpenetrans in less than a half cases and high percent of isolated injuries (42.60%). Most of them are from artillery projectiles.

Ticlopidine improves dialysis clearance of solutes in uremic patients by reducing blood clotting in dialyser fibers.

Ticlopidine, a platelet aggregation inhibitor was tested, in a double blind comparative cross-over study versus placebo, in 51 dialysed uremic patients who had increased dialyser blood clotting (> 25 fibers clotted/dialyser). At the end of a 7-day treatment period with 250 mg daily, the clearance of urea, creatinine and phosphate was determined at 30 and 210 minutes of dialysis, as well as the number of fibers clotted at the end of dialysis. Ticlopidine improved dialyser clearances for urea, creatinine and phosphate from 165 +/- 41 to 182 +/- 35 (p < 0.01), 135 +/- 37 to 143 +/- 35 (p < 0.05), and 120 +/- 36 to 130 +/- 35 (p < 0.05) ml/min, respectively, at 30 min of HD and a similar effect was seen after 210 min of dialysis. The number of dialyser fibers clotted after dialysis was reduced by ticlopidine therapy from 110 +/- 48 to 15 +/- 8 (p < 0.01). Ticlopidine reduced the initial dialysis-induced drop in leucocyte count by 20% (p < 0.05); no change in platelet or erythrocyte count was observed. Two out of 51 patients experienced an adverse reaction from ticlopidine (cutaneous haematoma and minor gingival bleeding). We conclude that ticlopidine is an efficient and safe drug for dialysed uremic patients since it can reduce blood clotting and thereby increase dialysis efficiency.

The role of sympathetic dysfunction in blood pressure regulation in uremic patients treated by continuous ambulatory peritoneal dialysis.

The role of autonomic nervous system dysfunction in the regulation of arterial blood pressure in 4 male and 10 female uremic patients, age 35-65 years (57 +/- 9 years), treated by continuous ambulatory peritoneal dialysis (CAPD) for 6-40 months (20 +/- 12 months), was studied. The integrity of the entire autonomic reflex arc was checked using Valsalva's test and the orthostasis challenge test. In parallel, efferent sympathetic function was tested with the handgrip test. A significantly decreased Valsalva ratio was found in 7 patients (50%), and it was normal in only 2 patients (14%). The handgrip test was subnormal in 5 patients (36%) and abnormal in 7 patients (50%). On the contrary, the orthostatic challenge test showed normal compensatory reaction in 10 patients (72%). The evaluation of three autonomic tests showed combined (afferent + efferent) lesion in 6 patients (43%), predominant afferent lesion in 3 patients (21%), and in 3 patients predominant efferent lesion. In 2 patients (14%) there was no end-organ response. Despite significant autonomic nervous system dysfunction in uremics on CAPD, as demonstrated by these results, blood pressure regulation in the supine and upright position was adequate in most patients (72%), indicating a relatively greater role of other blood pressure-regulating factors.

Phagocytic function in patients with recurrent urinary tract infections.

Phagocytes play an essential role in the host's defence against uropathogenic bacteria which are mostly extracellular pathogens. The functional capacity of peripheral blood phagocytes (predominantly polymorphonuclears, PMN) was investigated in 47 patients (urethrocystitis, acute or chronic pyelonephritis) and in healthy persons. The random mobility of phagocytes was determined by measuring their spontaneous migration from a capillary tube. Using radioactively labelled sheep erythrocytes as targets and the phagocytes as effector cells, ingestion, digestion and extracellular cytotoxicity were determined. All the four phagocytic functions in patients were significantly lower than in healthy controls, especially in patients with chronic pyelonephritis. These results link reduced phagocytosis by blood phagocytes with recurrent urinary tract infections (UTI). Whether the defect is primary or secondary to infection (and only transient) should be the object of further studies.

The effect of guanidino substances from uremic plasma on insulin binding to erythrocyte receptors in uremia.

We have studied insulin binding to erythrocyte receptors in a group of 25 nonobese, nondiabetic uremic patients undergoing maintenance hemodialysis for 2-54 months and 14 healthy controls. Erythrocytes of predialyzed uremics bind significantly less insulin than control erythrocytes (p less than 0.01). Dialysis resulted in a rapid increase of insulin binding (p less than 0.001). The concentrations of plasma insulin and glucose remained essentially unchanged during 5-hour hemodialysis and did not significantly differ from the control values. The down regulation of insulin receptors in undialyzed patients in the presence of normal plasma insulin concentration indicates that factors other than insulin itself could be responsible for insulin receptor activity during uremia. The results demonstrated that creatinine, creatine and glycocyamine have a direct suppressive effect on insulin binding of postdialyzed plasma (p less than 0.05) in concentration of 1 mmol/l. This suggested that specific uremic toxins could play an important role in the mechanisms of altered insulin binding during hemodialysis. Despite the high concentration of these compounds in blood of uremics, the only common feature for these compounds is the presence of the guanidino group in the molecule.