Novel RET mutations in macedonian patients with medullary thyroid carcinoma: genotype-phenotype correlations.

Medullary thyroid carcinomas (MTCs) are rare neoplasms comprising 2-10% of all thyroid malignnancies. More than 75% are sporadic tumors and the remainder is familial and MEN2 related. Both sporadic and syndromic MTCs frequently show mutations in the RET proto-oncogene. It has been noted that some MTC cases present an indolent, and some an aggressive clinical course. Ki-67 expression is generally low, with documented exceptions, whereas high expression of Bcl-2 has been reported in majority of the cases. Some studies have shown that Ki-67 and Bcl-2 expressions have prognostic value, as well as RET mutational status. We analyzed 20 unrelated MTC cases for Ki-67, Bcl-2 expression and RET mutations and tested their intercorrelations, correlations to the morphologic features and stage of the tumors, as well as their influence on survival. In 13 of the 20 analyzed cases we found 23 sequence changes distributed in exons 8, 10-13 and 16. There were 11 different missense mutations, single nucleotide deletion with frameshift, and 8 different synonymous mutations. Only 4 of the sequence changes have been previously published. Twelve patients (60%) had tumors expressing one or more missense mutations or single nucleotide deletion and 7 of them (35%) had at least one damaging or possibly damaging RET mutation. Most of the tumors had low Ki-67 expression (mean 6.48% of cells) and high Bcl-2 expression (mean 68.3%). Significantly better survival was observed in cases with low Ki-67 (< 6.5%; p < 0.05), high Bcl-2 expression (> 68.3%; p < 0.01) and younger age at diagnosis (< 51 years; p < 0.05).

Sentinel lymph node detection in breast cancer - first experience.

INTRODUCTION: Breast cancer accouns for 22.9% of all cancers in women and 13.7% of cancer deaths. Positive axillary lymphnodes (ALN) predict the development of distant metastases. The status of the sentinel lymphnode (SLN) is crutial for the treatment selection. AIM: To determine the benefits of SLN detection in patients with breast cancer. MATERIAL AND METHODOLOGY: 38 female patients (pts), age 44 ± 12 years, with T1-2 N0 M0 breast cancer, without enlarged ALN on ultrasound (US), were included. SLN detection was performed using gamma camera and gamma detection probe after periareolar subcutaneous and/or peritumoral injection of (99m-Technetium-SENTISCINT). Blue dye was administered 20 min before the operation. SLN was extirpated and ex tempore histopathology was performed. RESULTS: Ex tempore SLN evaluation was negative and the lymphatic pathways preserved in 28/38 (74%) pts. In 10/38 (26%) pts SLN was positive, followed by radical surgery. In 3/28 ex tempore negative patients, histopathological analysis showed metastatic involvement (false negative). In 3/10 ex tempore positive patients micro metastases 0,2-2 mm were detected. 12 pts had 2 SLN, 8/12 (66%) had negative and 4/12 (34%) had positive SLN. 3 pts had a rare double drainage to axilla and a. mammaria int. CONCLUSION: Our results confirm that SLN detection technique is non-invasive, safe and reliable and should be incorporated into the guidelines for breast cancer pts (T1-2 N0 M0). The most reliable option for colloid application is the combined technique of periareolar and peritumoral injection. Patients with drainage to a. mammaria interna should be selected for adjuvant protocols.

Perindopril treatment in streptozotocin induced diabetic nephropaty.

Diabetic nephropathy (DN) is one of the most common causes of terminal stadium damage to the kidneys. The angiotensin-converting enzyme (ACE) represents a significant risk factor for the progression of DN. ACE inhibitors are medications of particular interest knowing the role of angiotensin II in the development of DN. This study aimed to examine the effects of ACE inhibitor treatment perindopril (PER), administered to rats with streptozotocin (STZ) induced DN, that developed albuminuria, renal hypertrophy and mild glomerulussclerosis. DN was induced by a STZ (60 mg/kg ip) single injection to normotensive Wistar rats. The administration of STZ caused diabetes mellitus (DM) with symptoms and signs of DN including poor general condition, body-weight loss, kidney weight increase as well as increased values of BUN and serum creatinine, accompanied by increased diuresis as well as distinct albuminuria. The majority of these symptoms were manifested 4 weeks after, and even more distinctly 8 and 12 weeks after administering STZ. The perindopril treatment (6 mg/kg BW), starting 4 weeks after administering STZ, resulted in a significant improvement of all symptoms and signs of DN, significantly lowering the values of BUN and serum creatinine, albuminuria and diuresis. The histopathological examination of the renal samples at 8 and 12 weeks after the beginning of the study have shown that perindopril significantly lowers the progression of glomerulopathy, and significantly improves the glomerulosclerotic index, as well as the progression of renal histological abnormalities induced with STZ. Thus perindopril treatment ameliorates STZ-induced nephropathic changes in DM rats.

Proteomic analysis of infiltrating ductal carcinoma tissues by coupled 2-D DIGE/MS/MS analysis.

There is a growing interest in protein expression profiling aiming to identify novel diagnostic markers in breast cancer. Proteomic approaches such as two-dimensional differential gel electrophoresis coupled with tandem mass spectrometry analysis (2-D DIGE/MS/MS) have been used successfully for the identification of candidate biomarkers for screening, diagnosis, prognosis and monitoring of treatment response in various types of cancer. Identifying previously unknown proteins of potential clinical relevance will ultimately help in reaching effective ways to manage the disease. We analyzed breast cancer tissues from five tumor and five normal tissue samples from ten breast cancer subjects with infiltrating ductal carcinoma (IDC) by 2-D DIGE using two types of immobilized pH gradient (IPG) strips: pH 3-10 and pH 4-7. From all the spots detected, differentially expressed (p < 0.05 and ratio > 2) were 50 spots. Of these, 39 proteins were successfully identified by MS, representing 29 different proteins. Ten proteins were overexpressed in the tumor samples. The 2-D DIGE/MS/MS analysis revealed an increase in the expression levels in tumor samples of several proteins not previously associated with breast cancer, such as: macrophage-capping protein (CAPG), phosphomannomutase 2 (PMM2), ATPase ASN1, methylthioribose-1-phosphate isomerase (MRI1), peptidyl-prolyl cis-trans isomerase FKBP4, cellular retinoic acid-binding protein 2 (CRABP2), lamin B1 and keratin, type II cytoskeletal 8 (KRT8). Ingenuity Pathway Analysis (IPA) revealed highly significant (p = 10(-26)) interactions between the identified proteins and their association with cancer. These proteins are involved in many diverse pathways and have established roles in cellular metabolism. It remains the goal of future work to test the suitability of the identified proteins in samples of larger and independent patient groups.

Histological features, Ki-67 and Bcl-2 immunohistochemical expression and their correlation with the aggressiveness of pheochromocytomas.

Pheochromocytomas and paragangliomas are rare neoplasms. Approximately 10% may present malignant behaviour. There are no reliable morphological signs of malignancy, except for the presence of metastasis. We performed morphological and immunohistochemical analysis on 15 pheochromocytomas and 5 paragangliomas aiming to find correlations between the morphological features of the tumours, immunohistochemical expressions of Ki-67 and Bcl-2, and the biological behaviour of the tumours. According to the biological behaviour of the tumors, the patients were divided into an indolent disease group (ID), and an aggressive disease group (AD). The morphological analysis included the PASS core parameters, greatest tumour diameter and weight, as well as age and gender of the patients, survival and disease-free periods after operation. According to histomorphological parameters, tumours were divided into tumours with "benign-like" morphology and tumours with "malignant-like" appearance. The disease course was neither correlated to the PASS score, nor to the individual parameters comprising it. The rest of the morphological parameters and the immunohistochemical expressions of Ki-67 and Bcl-2 were not able to predict the disease course, although we found significantly higher Ki-67 expression in paragangliomas in comparison to pheochromocytomas (p<0.01). Some of the PASS parameters (vascular invasion and presence of atypical mitoses) were positively correlated to the tumour weight (R=0.75; p<0.01, and R=0.56; p<0.05, respectively). The disease course was in positive correlation to the tumour weight, presence of vascular invasion and atypical mitoses; however there were no statistically significant differences regarding those parameters between the ID and AD groups (p>0.05).

MicroRNAs in Breast Cancer -Our Initial Results.

MicroRNAs (miRNAs) are small [∼21 nucleotide (nt)] non coding RNAs (ncRNAs) that regulate gene expression posttranscriptionally. About 3.0% of human genes encode for miRNAs, and up to 30.0% of human protein coding genes may be regulated by miRNAs. Currently, more than 2000 unique human mature microRNAs are known. MicroRNAs play a key role in diverse biological processes including development, cell proliferation, differentiation and apoptosis. These processes are commonly dysregulated in cancer, implicating miRNAs in carcinogenesis, where they act as tumor supressors or oncogenes. Several miRNAs are associated with breast cancer. Here we present our initial results of miRNA analyses of breast cancer tissues using quantitative real time-polymerase chain reaction (ReTi-PCR) (qPCR) involving stem-loop reverse transcriptase (RT) primers combined with TaqMan® PCR and miRNA microarray analysis.

Renal histopathology and clinical course in patients with Wegener's granulomatosis--single centre experience from the Republic of Macedonia.

The aim of this study was to evaluate the clinical course of patients with Wegener's granulomatosis (WG) with renal involvement, to examine histopatological form seen in renal biopsies and present follow-up of the patients. A retrospective analysis was carried out of 18 patients presenting with WG and active renal disease at the University Nephrology Department, Ss. Cyril and Methodius University, Skopje, R. Macedonia. All patients were ANCA positive and had a percutaneous renal biopsy taken on their admission. 12 patients were male, 6 female, aged 48.61±13.77 (M±SD). All had extrarenal symptoms prior to admission. Oligoanuria was present in 7/18 (38.9%) of the patients, serum urea levels of the whole group were 40.67±18.13 mmol/l (M±SD) and for serum creatinine 691.06±384.93 µmol/l (M±SD). Necrotizing glomerulonephritis with crescents was present in 11/18 (61.11%) of the patients, the others presented diffuse proliferative extracapillary glomerulonephritis. All patients were treated with steroids and cyclophosphamide, and plasmapheresis was performed in 7/18 (38.9%) of the patients. Probability rate for surviving after one month was 0.6111 and after three months 0.3889 (Kaplan-Meier). The current treatment of WG in our study did not prevent serious complications and development of ESRD in a large number of our patients. This systemic disorder is still a serious problem and early diagnosis and alternative strategies for the management of the disease will be an important objective for further studies.

Immunosupressive treatment with cyclosporin-A for a patient with hypoplastic myelodisplastic syndrome: a case report.

BACKGRAOUND: Imunosupressive therapy with antithymocyte globulin (ATG), cyclosporine (CsA) or both has been shown to induce haematological responses in a subset of patients with myelodysplastic syndromes (MDS), in particular in the hypocellular form of MDS. CASE REPORT: We report our first case with hypocellular MDS treated with CsA. A 54-year-old female referred to our Department due to weakness and severe pancytopenia. Hypocellular form of MDS was diagnosed after bone marrow biopsy. Treatment with CsA was started one year after diagnosis. Treatment with CsA resulted in clinical improvement, a very good partial haematological response, resolution of transfusion requirement and an increase in bone marrow cellularity. CONCLUSIONS: In our experience, immunosuppressive treatment with CsA and/or ATG could be an alternative for patients with hypoplastic MDS for whom there is no possibility of allogenic bone marrow transplantation as only curative therapy.

Dose-dependent therapeutic effects of 2-Methoxyestradiol on Monocrotaline-Induced pulmonary hypertension and vascular remodelling.

2-Methoxyestradiol (2ME) is a major non-oestrogenic metabolite of oestradiol. Our previous studies, performed in several models of cardiac and/or vascular injury, suggest that 2ME strongly inhibits both pressure-dependent and pressure-independent cardiac and vascular remodelling. Furthermore, recently we have shown that in male rats 2ME attenuates the development and retards the progression of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH); and in female rats 2ME eliminates the exacerbation of PAH and increased mortality due to ovariectomy. In the present study we compared the therapeutic effects of three different doses of 2ME (3, 10 and 30 microg/kg/hour; 2ME-3, 2ME-10 and 2ME-30, respectively) in male rats with MCT-induced PAH. The animals were also monitored for plasma 2ME levels and potential oestrogenic effects. Treatments were initiated 12 days after administration of MCT (60 mg/kg, i.p.). Twenty-eight days post MCT, right ventricular peak systolic pressure (RVPSP) was measured and morphometric analysis was conducted. All three doses of 2ME produced beneficial therapeutic effects in pulmonary hypertensive animals, i.e. reduced pulmonary artery pressure and right ventricular hypertrophy, attenuated pulmonary vascular remodelling and inflammatory response, and had favourable effects on survival. Notably, none of the three doses had any effect on plasma testosterone levels or on seminal vesicle or testicle weight. Dose-dependent increases in 2ME plasma levels were observed only with 2ME-3 and 2ME-10; 2ME-30 produced 2ME plasma levels similar to those seen with 2ME10. Nonetheless, 2ME-30 was significantly more efficacious than 2ME-3 or 2ME-10 and eliminated the high mortality (34%) induced by MCT. In summary, the present study indicates that 2ME, used in doses that produce plasma levels similar to those seen in the last trimester of pregnancy (1000-3000 pg/ml), is effective and safe (i.e. has no oestrogenic effects) in experimental PAH. These data also suggest that 2ME disposition, rather than plasma concentration, determines the therapeutic effects of 2ME in PAH.

Bioefficacy of budesonide loaded crosslinked polyelectrolyte microparticles in rat model of induced colitis.

A targeted delivery system for inflammatory bowel diseases, chitosan-Ca-alginate microparticles efficiently loaded with budesonide (BDS), were designed using one-step spray-drying process. They were eudragit-coated and examined for in vivo efficacy. Experimental colitis was induced by rectal instillation of 2,4,6-trinitrobenzene sulphonic acid (TNBS) into male Wistar rats. Drugs were administered by oral gavage daily for 5 days. Colon/body weight ratio, gross morphological and histological evaluation, and clinical activity score were determined as inflammatory indices. Individual clinical and histological evaluation showed that colitis severity was suppressed the most greatly in order BDS < BDS/C-Ca-A < E-BDS/C-Ca-A. Clinical activity score decreased in the same order. Statistical analyses of total score points indicate that the incorporation of BDS in microparticles had significant differences in favor of efficacy of designed delivery system with mucoadhesive and controlled release properties (one-way ANOVA, P < 0.05). The results established the prediction by previous in vitro studies.

Progesterone inhibits vascular remodeling and attenuates monocrotaline-induced pulmonary hypertension in estrogen-deficient rats.

(Full text is available at http://www.manu.edu.mk/prilozi). Pulmonary arterial hypertension (PH) is predominantly a disease of young females. Yet, little is known regarding the effects of female sex hormones in PH. Female rats develop less severe PH compared to male rats, and ovariectomy (OVX) exacerbates PH. Although OVX rats treated with estradiol develop less severe disease, the role of progesterone in OVX-induced exacerbation of disease has not been examined. Progesterone was shown to dilate pulmonary vessels and to inhibit proliferation of endothelial and vascular smooth muscle cells. Therefore, we hypothesized that progesterone may confer protective effects in experimental PH. A total of 30 female rats were ovariectomized and OVX rats were randomly administered either saline (OVX-Control group, n = 7), monocrotaline (60mg/kg i.p.; OVX-MCT group; n = 12), or MCT plus progesterone (30microg/kg/h via osmotic minipumps; OVX-MCT+P group; n = 11). After 32 days animals were instrumented for in situ (open chest) measurements of right ventricle (RV) peak systolic (RVSP) and end diastolic (RVEDP) pressures, and tissue samples were obtained for morphometric and histological analysis. Administration of MCT elevated RVSP (22.2 +/- 1.1 vs. 46.7 +/- 2.4 mmHg) and RVEDP (1.51 +/- 0.86 vs. 11.9+/-2.2 mmHg), increased RV/left ventricle + septum (RV/LV+S) ratio (0.256 +/- 0.010 vs. 0.582 +/- 0.033, OVX vs. OVX-MCT), and induced media hypertrophy of small size pulmonary arteries. In ovariectomized pulmonary hypertensive rats, treatment with progesterone attenuated the severity of disease (OVX-MCT+P group: RVSP = 36.6 +/- 2.3 mmHg; RV/LV+S = 0.468 +/- 0.025; RVEDP = 7.5 +/-1.5 mmHg), attenuated vascular remodeling (media % index: 28.2 +/- 1.1 vs. 34.2 +/- 1.3), and reduced mortality (9% vs. 25%; OVX-MCT+P vs. OVX-MCT). This study provides the first evidence that in estrogen-deficient rats, progesterone has protective effects in MCT-induced PH. Further evaluation of the role of progesterone and its interaction with estrogens in pulmonary hypertension is warranted. Key words: Pulmonary Hypertension, Progesterone, Estrogens, Vascular remodeling.

Erythropoietin reduces cumulative nephrotoxicity from cisplatin and enhances renal tubular cell proliferation.

Cisplatin, a heavy metal complex, is one of the most active drugs used in the treatment of several human malignancies. However, high-dose therapy with cisplatin is limited by its cumulative nephrotoxicity. The main objectives of this study were to determine the role of recombinant human erythropoietin (Epoetin alfa) in the prevention of nephrotoxicity induced experimentally in Wistar rats by long-term administration of cisplatin (2 mg/kg/b.w./week) over eight weeks, and an evaluation of its effect on renal tubular cell proliferation. The animals were randomly assigned into three groups, each including 25 rats. Group 1 (CP) received only cisplatin (2 mg/kg/b.w./week), group 2 (CP+EPO) received cisplatin (2 mg/kg/b.w./week) and epoetin alfa (150 IE/kg/b.w./three times a week), and group 3 (control group) received only saline. During the study, the following tests for the assessment of the renal function and renal damages were performed: determination of concentration of serum creatinine and BUN and determination of total protein quantity in 24-hour urine samples. At the end of the study, the abdomen was opened and both kidneys of the rats were removed and sent for histological and morphometric analysis. Ki-67 was used as a tool to determine a proliferative index. The results obtained have shown that epoetin alfa significantly reduced the functional renal failures and renal damages, and increased toleration of high doses of cisplatin. At the same time, our results with regard to tubular proliferative index have confirmed that one of the possible mechanisms by which erythropoietin accomplishes its renoprotective effect is stimulation of tubular cell proliferation and regeneration.

Bcl-2 as a prognostic factor for survival in small-cell lung cancer.

BACKGROUND: The expression of Bcl-2 oncoprotein is associated with inhibition of apoptosis and prolonged cell survival. The purpose of this study was to investigate Bcl-2 protein expression in patients with small-cell lung cancer (SCLC) in order to see if it was related to clinicopathological features and to prognosis. MATERIALS AND METHODS: Forty patients with SCLC were stained immunohistochemically using specific monoclonal antibody (DAKO-Bcl-2, 124). Bcl-2 positivity was determined as detection of the oncoprotein in greater than 10% of neoplastic cells. RESULTS: Immunopositivity was present in 26 (60%) of SCLC patients. Twenty-three of 40 (57.5%) patients had limited disease at presentation, and 17 of 40 (42.5%) had extensive disease. There was not any correlation with Bcl-2 protein expression and clinicopathological parameters such as sex, age, smoking history and performance status. According to the extent of the disease, Bcl-2 expression was significantly higher in patients with extensive disease (p < 0.009). Bcl-2 expression was associated with significant shorter survival in patients with SCLC (Log Rank = -5.26; p = 0.00001). Cox regression analysis controlling for age, sex and tumor stage, confirmed that Bcl-2 expression (HR = 0.049 p < 0.0001) and N stage (HR = 0.152 p < 0.012) were an independent prognostic markers for poor prognosis. In CONCLUSION Bcl-2 oncoprotein was expressed in most cases of SCLC and its expression may have prognostic importance.

Rituximab in the treatment of the variant of hairy cell leukaemia: a case report.

Hairy cell leukaemia (HCL) is an uncommon, low-grade B-cell lymphoproliferative disorder. HCL-variant describes an entity of HCL that is important from the point of view of requiring differential diagnosis from HCL, and for requiring careful consideration of the treatment approach. HCL-variant differs from the classic form with respect to the lack of monocytopaenia, its elevated WBC and unique morphology and immunophenotype. Indeed, there is currently no adequate standard treatment for this condition - HCL-variant is generally resistant to interferon-alpha, and complete remission is rarely achieved with either pentostatin or cladribine. We report a 57-year-old female patient who presented at our institution in November 2004 with high white blood counts and splenomegaly. Based on her blood morphology, bone marrow and spleen histology, immunophenotype and clinical characteristics, the patient was diagnosed as having HCL-variant, with blastoid variant transformation. The patient had advanced-stage disease. She was initially treated with spleenctomy, which resulted in short-term normalization of blood counts. One month later the blood counts deteriorated, she developed peripheral and abdominal lymphadenopathy and had poor performance status. One cycle of cladribine combined with rituximab was immediately administered. We started with rituximab 375 mg/m(2), which resulted in a remarkable recovery of blood counts, followed by cladribine 0.1 mg/kg for 7 days. However, the patient's general condition worsened, and she subsequently died from heart failure. Our experience from this case suggests that rituximab is a promising therapy for patients with HCL-variant, particularly when combined with cladribine. However, further clinical study is required before rituximab can be considered as a front-line therapy for this form of malignancy.

Non-Hodgkin's lymphomas: immunologic prognostic studies.

Non-Hodgkin's lymphomas are malignant lymphoproliferative disorders that originate from B or T-lymphocytes and rarely from NK cells. They represent an extremely heterogeneous group of diseases regarding histologic subtypes, clinical presentations, immunophenotypic profiles, cytogenetic and molecular features and suitable mode of treatment. A clinical indicator of prognosis, the International Prognostic Index, takes into account factors that are mostly linked to patient characteristics (age, performance status) and to disease extension and growth (disease stage, s.lactate dehydrogenase level and extent of extranodal involvement). However, it is clear that differences in clinical features and in treatment responses are a result of the marked genetic, immunophenotypic and molecular heterogeneity that underlie disease aggressiveness and tumour progresssion. We applied IPI (based on pretreatment clinical characteristics) in our group of 136 patients that identified a subgroup of clinical features that remained independently significant in multivariate analyses. In our results IPI turned out to be of prognostic significance for response rate and survival percentages. Based on their number of "poor risk" factors, patients were placed into four IPI risk groups: low (one or no risk factors), low-intermediate (two risk factors), intermediate-high (three risk factors), and high (four or five risk factors) with five years survival rates of 88%; 82%; 18% and 0% respectively. However, one limitation of this prediction strategy is that IPI does not encompass molecular abnormalities of tumour cells, which may play a critical role in determining profoundly different clinical outcomes in patients within the same group as defined by IPI. The aim of this study was to assess the clinical significance and potential prognostic value of the expression of the new immunologic prognostic markers including nuclear proliferating antigen, suppressor and oncogenic proteins, HLA-DR surface antigens, tumour infiltrating T-lymphocytes, lymphocyte homing receptor and angiogenic molecules. Immunohistochemistry was used to examine paraffin-embedded tumour tissues for determining the expression of immunologic prognostic markers. Survival analysis showed that serum-high lactate dehydrogenase level, poor performance status (ECOG 3, 4 and 5), high proliferative activity defined as nuclear Ki67 expression greater than 60% of malignant cells and high tumour invasive potential defined by discontinued or loss of Collagen IV were found as strong predictors of poor survival among these patients. These four prognostic parameters determined the New-PI with three risk groups: good (0-1 risk factors), intermediate (2 risk factors) and poor (3 and more risk factors), with predicted five-years survival rates of 88%, 64% and 0%. The New-PI more accurately predicts the outcome than the standard IPI (p < 0,001 vs. p < 0.0001). Based on a single institution series of 136 patients the IPI has proved to be a useful prognostic tool for NHL patients. Addition of new cellular markers into the standard IPI significantly improves risk stratification in NHL.

Do we have to treat subclinical rejections in early protocol renal allograft biopsies?

The aim of the present study was to evaluate whether treatment of subclinical, borderline rejections (SR/BR) or histological findings of chronic allograft nephropathy (CAN) in protocol biopsies in the first month posttransplantation after living related kidney transplantation has a beneficial effect on graft histology and renal function at 6 months. Among the 40 paired biopsies, only 6/80 showed no histological lesions. BR was found in 13/40 and 12/40, and SR in 15/40 and 21/40 of patients on the 1- and 6-month biopsies, respectively. The mean histological index/total sum of scores for acute and chronic changes (HI) increased at 6-month biopsy: 5.3 +/- 2.9 vs 7.8 +/- 3.6 (P < .001). Similarly, the mean sum of histological markers for chronicity (CAN score) of 2.1 +/- 1.5 increased to 4.6 +/- 2.3 (P < .001) on the 6-month biopsy. When divided according to whether there was treatment of BR and SR, the treated BR/SR group on 1-month biopsy had a mean HI score of 7.11 +/- 1.9, which remained almost the same (7.11 +/- 2.32) at 6 months. Among the untreated BR/SR group it increased from 4.95 +/- 1.99 to 8.16 +/- 4.30. However, there was no difference in graft function between the groups from 1 to 6 months. In conclusion, a protocol 1-month biopsy may be valuable to establish the prevalence of BR/SR in stable allografts. The presence of an untreated BR/SR upon a 1-month biopsy showed greater susceptibility for histological deterioration on the 6-month biopsy due to an accelerated CAN process.

Morphological changes in the tubulointerstitial compartment in primary glomerulopathies.

The renal interstitium structurally supports the functional renal units and is involved in almost all renal functions. The degree of renal disfunction strongly correlates to the changes in the tubulointerstitial compartment present in almost all types of glomerular diseases. A phenomenon arising in such an environment is epithelial-mesenchymal transition, i.e. a change of the cell;s epithelial phenotype into a mesenchymal one. Histochemical, immunohistochemical and morphometric analyses were made of 50 renal biopsies with primary glomerulopathies, as well as light-microscopy analyses of semi-thin sections embedded in epoxy resin. Double immunohistochemical stainings with pairs of epithelial and mesenchymal antibodies were also done. The results were analyzed and correlated with the clinical data of the renal function of the patients. The immunohistochemical analyses of the atrophic tubular epithelial cells showed a loss of expression of Cytokeratin and E-cadherin, an enhanced expression of HLA-DRalpha, and a de novo expression of Vimentin and alphaSMA as markers for epithelial-mesenchymal transition. The double immunohistochemical stainings with Cytokeratin/Vimentin and Cytokeratin/alphaSMA showed a simultaneous expression of these antigens in atrophic tubular cells. Their proliferative index was mildly enhanced. Interstitial fibrosis was present in 98% of the analysed biopsies. The analyses show correlations among all the changes in the tubulointerstitial compartment as well as the concentration of creatinine in the serum as a parameter of renal function. The study emphasizes the usefulness of the implementation of histomorphometrical and immunohistochemical techniques as well as ultrastructural and molecular analyses in the process of nephropathological diagnosis.

Evaluation of telomerase activity in patients with chronic B lymphocytic leukemia versus age matched controls. Correlation between telomerase activity and bone marrow infiltration.

Telomerase is a ribonucleoproteic enzyme associated with cellular immortality and malignancy. This enzyme, besides the catalytic subunit bearing reverse transctiptase activity, contains an RNA template complementary to TTAGGG telomeric repeats, thus permitting de novo synthesis of telomeric DNA onto chromosomal telomeric ends. Increased telomerase activity has been reported in Chronic Lymphocytic Leukemia (CLL) by many authors. In order to investigate the telomerase activity in patients with CLL and its correlation to commonly used morphologic prognostic markers, 38 frozen blood lymphocyte samples from patients with CLL and 47 age-matched controls were investigated for telomerase activity using the Telomerase PCR ELISA-plus kit from Roche. Trepanobiopsies from the same patients were analysed for the type of bone marrow infiltration as well. Analysis showed highly variable Relative Telomerase Activity (RTA) in B-CLL patients, ranging from comparable or even lower than the mean RTA of controls (in Binet A stage patients) to manifold increase in the majority of patients with advanced stage disease. The sex and age of the patients showed no influence on RTA in CLL patients, in contrast to the control group, where the age influenced telomerase activity. We found a positive correlation between the RTA and disease stages (Binet), as well as between RTA and the type of BM infiltration.

ANCA-GBM dot-blot test in diagnosis of patients with glomerulonephritis.

Patients with rapidly progressive glomerulonephritis who are positive for anti-neutrophil cytoplasmic antibody (ANCA) or anti-glomerular basement membrane (GBM) antibodies may develop chronic renal failure leading to end-stage renal disease (ESRD) within days or weeks. The early serologic detection of auto-antibodies associated with ANCA and anti-GBM diseases will be helpful in preventing ESRD. We evaluated the combined ANCA-GBM dot-blot strip assay (Biomedical Diagnostics, Brugge, Belgium) in 30 consecutive patients with biopsy proven glomerulonephritis (GN). MPO- and PR3-ANCA were detected in 5 and 2 samples, respectively. Three samples were positive for both MPO- and PR3-ANCA (all 3 had focal segmental necrotizing GN). One patient was diagnosed as having Goodpastures' syndrome (the only anti-GBM positive result) and two had Wegener's granulomatosis (the two PR3-ANCA positive results). Two additional samples were equivocal: positive for MPO-ANCA and PR3-ANCA, respectively. Patients positive only for MPO-ANCA had only limited extrarenal organ manifestations. Anti-PR3 positive patients with necrotizing glomerulonephritis had a more dramatic deterioration of their renal function at diagnosis. Radiographically, these patients had nodular or pneumonia-like lesions. Acute respiratory failure necessitating mechanical ventilation was developed in one GBM positive patient. In conclusion, the ANCA-GBM dot-blot is a useful screening tool in situations where conventional ANCA testing is not readily available.