SPR imaging biosensor for determination of laminin-5 as a potential cancer marker in biological material.

A new method for the selective determination of laminin-5 concentration using a biosensor and surface plasmon resonance imaging (SPRI) technique is presented. A biosensor based on the specific interaction of laminin-5 with rabbit polyclonal antibody was constructed. The analytically useful dynamic response range of the biosensor is between 0.014 and 0.1 ng mL(-1). The detection limit is 4 pg mL(-1). The potential influence of interferences on the SPRI signal was investigated, and the high selectivity of the biosensor was confirmed. In order to demonstrate the potential application of the biosensor, laminin-5 concentration in blood plasma was determined. The results were compared with the laminin-5 concentration obtained by the commercial enzyme-linked immunosorbent assay (ELISA) kit. A comparison of results from healthy donors obtained by SPRI measurement and ELISA indicates that they are close and shows good agreement with the data reported in the literature. The plasma samples of bladder cancer patients gave higher concentration measured with specific biosensor than by ELISA assay. The study shows the clear difference in concentration of laminin-5 in healthy humans and patients with bladder cancer. Extensive clinical studies using the newly developed method can result in an increase in the use of laminin-5 as a potential cancer marker.

Carbohydrate-deficient transferrin depends on disease activity in rheumatoid arthritis and systemic sclerosis.

OBJECTIVES: Changes in the glycosylation of plasma proteins have been linked to the aetiology of the rheumatic diseases. The aim of this study was to determine and compare the levels of carbohydrate-deficient transferrin (CDT) in patients with rheumatoid arthritis (RA), systemic sclerosis (SSc), and systemic lupus erythematosus (SLE). METHOD: Studies were carried out in 29 female patients with RA, 27 with SSc, and 17 with SLE. CDT was assayed by the N Latex CDT immunonephelometric assay. RESULTS: The levels of %CDT in the sera of RA, SLE, and SSc patients were significantly higher than in controls while the absolute concentrations of CDT were unchanged. %CDT, CDT, and transferrin do not differ significantly between patients with rheumatic diseases. In RA and SSc patients, a positive correlation was observed between %CDT and C-reactive protein (CRP), as well as a positive correlation in RA patients between %CDT and 28-joint Disease Activity Score (DAS28). CONCLUSIONS: The changes in the serum %CDT concentration in patients with RA and SSc correlated with disease activity markers.

Relationship between CDT and disease activity in rheumatoid arthritis.

The aim of this study was to estimate the serum concentration of carbohydrate-deficient transferrin (CDT) in patients with rheumatoid arthritis (RA) and the relationship between the CDT level and disease activity in RA patients. Studies were carried out in 47 female patients with RA and 32 healthy women. Disease activity of RA was evaluated using the 28-joint count Disease Activity Score (DAS 28). Serum CDT was determined by particle-enhanced immunononephelometry using the N Latex CDT test. Patients with RA had significantly lower serum concentrations of CDT compared with controls. The correlation study showed the significant negative relationship between CDT and DAS 28 (r = - 0.483, p = 0.011). There were no correlations between serum CDT level and patient's age, disease duration, number of tender and swollen joints, and degree of disability evaluated by the Health Assessment Questionnaire. The level of CDT in patients with RA was significantly decreased and confirms the changes in transferrin glycosylation which are dependent on the disease activity. Therefore, measurement of CDT in the sera of patients with RA can be useful for the evaluation of disease activity in these patients.

Relationship between serum acute-phase proteins and high disease activity in patients with rheumatoid arthritis.

PURPOSE: The aim of this study was to assess the relationship between serum acute-phase proteins and high disease activity evaluated by activity score (DAS28) in patients with rheumatoid arthritis. MATERIAL/METHODS: Studies were carried out on 27 females with RA and 32 control women. Acute-phase proteins were divided into 4 fractions as follows: alpha1-globulins represented by alpha1-acid glycoprotein (AGP) and alpha1-antitrypsin (AAT); alpha2-globulins - haptoglobin (Hp); beta-globulins - complement C3 (C3) and total transferrin (Tf); gamma-globulins - C reactive protein (CRP), rheumatoid factor (RF) and immunoglobulin G (IgG), and determined by immunoturbidimetric methods. RESULTS: The serum levels of acute-phase proteins changed in RA patients. On account of the alterations of concentration, acute-phase proteins are placed in the downgrade scale as follows: CRP, Hp, AGP, C3, AAT and Tf. None of the acute-phase proteins correlated with the RF and the majority of them were closely related to ESR. Almost all of the acute-phase proteins (without C3) were closely related to RA activity (based on DAS28) and their places in the downgrade scale were as follows: CRP, Tf, AGP, Hp and AAT. The degree of disability evaluated by Health Assessment Questionnaire has affected on the concentrations of AGP, Tf and CRP. Serum AGP, AAT and RF levels significantly correlated with the patient's age. No correlations were observed between IgG, TP levels, and clinical data. CONCLUSIONS: Among the entire panel, the CRP and AGP appeared to be the most useful biochemical markers for evaluation of the disease activity of patients with RA.

Carbohydrate-deficient isoforms of transferrin (%CDT) and sialic acid (SA) in iron-deficiency anemia.

This study has investigated the serum levels of carbohydrate-deficient isoforms of transferrin (CDT) and sialic acid (SA) in iron-deficiency anemia (IDA). Blood samples were collected from 60 women with IDA and from 20 healthy controls. CDT was estimated by anion-exchange chromatography on minicolumns followed by photometric detection of transferrin and was expressed as a percentage of total transferrin (%CDT). SA was measured by an enzymatic method. There was no difference in the mean level of %CDT between patients with IDA (2.26%) and control patients (2.05%). SA increased significantly from control level 0.61 to 0.69 g/l in anemic patients. We concluded that elevated concentration of total transferrin in IDA did not change the relative value of low sialylated isoforms (%CDT) and the increase of total SA level in the sera of anemic patients is not related to the increase of total transferrin.

Interleukin-6, soluble interleukin-2 receptor and soluble interleukin-6 receptor in the sera of patients with different histological patterns of rheumatoid synovitis.

OBJECTIVE: The present study was conducted to investigate whether the serum levels of interleukin 6 (IL-6), soluble IL-2 receptor (sIL-2R) and sIL-6R are associated with the morphological appearance of rheumatoid arthritis (RA). METHODS: Using the ELISA technique we measured the IL-6, sIL-2R and sIL-6R concentrations in the serum of 34 patients with RA and 28 patients with osteoarthritis (OA). Histological analysis of synovial samples distinguished 2 types of rheumatoid synovitis. Twenty-one RA specimens presented diffuse infiltrates of mononuclear cells without any specific microanatomical organization. In remaining 13 samples the formation of lymphocytic follicles with germinal center-like structures was found. RESULTS: Serum levels of IL-6, sIL-2R and sIL-6R were elevated in patients with RA compared to the OA control group (p < 0.001, p < 0.001 and p < 0.05 respectively). Concentrations of IL-6 and sIL-2R were highest in the serum of RA patients with follicular synovitis in comparison to patients with diffuse synovitis (p < 0.001 and p < 0.01 respectively) and could distinguish RA patients with these two histological variants of the disease. Serum levels of IL-6 and sIL-2R correlated with markers of disease activity such as ESR and CRP levels. In addition, the clinical data suggest a more severe disease among RA patients with follicular synovitis. CONCLUSION: Distinct histological types of rheumatoid synovitis associated with unique serum concentrations of IL-6 and sIL-2R reflect levels of disease activity and confirm the concept of RA heterogeneity.

Circulating tumour necrosis factor alpha and soluble tumour necrosis factor receptors in patients with different patterns of rheumatoid synovitis.

OBJECTIVE: To examine the relation between the serum levels of tumour necrosis factor alpha (TNFalpha), soluble tumour necrosis factor receptors (sTNF-R), and the histological pattern of rheumatoid synovitis. METHODS: An enzyme linked immunosorbent assay (ELISA) was used to measure TNFalpha, p55 sTNF-R, and p75 sTNF-R concentrations in the serum of 43 patients with rheumatoid arthritis (RA) and 34 patients with osteoarthritis (OA). RESULTS: Upon histological analysis two variants of rheumatoid synovitis emerged. Twenty six RA specimens presented only diffuse infiltrates of mononuclear cells. In the remaining 17 samples the formation of lymphocytic follicles with germinal centre-like structures was found. Serum concentrations of TNFalpha, p55 and p75 sTNF-R were raised in patients with RA compared with the OA control group (p<0.001 for all comparisons). Levels of TNFalpha, p55 and p75 sTNF-R were higher in the serum of patients with RA with follicular synovitis than in patients with diffuse synovitis (p<0.001, p<0.01, and p<0.05, respectively). Serum concentrations of TNFalpha, p55 and p75 sTNF-R correlated with markers of disease activity. CONCLUSION: Different histological types of rheumatoid synovitis associated with distinct serum levels of TNFalpha and sTNF-R reflect varying clinical activity of the disease and support the concept of RA heterogeneity.

Soluble adhesion molecules (ICAM-1, VCAM-1, and E-selectin) and vascular endothelial growth factor (VEGF) in patients with distinct variants of rheumatoid synovitis.

BACKGROUND: Cell adhesion molecules and endothelial growth factors have an important role in the infiltrating of rheumatoid synovium with mononuclear cells, leading to the initiation and progression of the disease. OBJECTIVE: To investigate whether the serum profile of soluble adhesion molecules and of vascular endothelial growth factor (VEGF) is associated with the histological appearance of rheumatoid arthritis (RA). METHODS: Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-selectin), and VEGF were assessed by enzyme linked immunosorbent assay (ELISA) in 40 patients with RA and 32 patients with osteoarthritis (OA). RESULTS: Histological analysis of synovium specimens distinguished two types of rheumatoid synovitis. Twenty four RA samples presented diffuse infiltrates of mononuclear cells without any further microanatomical organisation, whereas in the remaining 16 samples lymphocytic follicles with germinal centre-like structures were identified. In comparison with patients with OA, constituting a control group, higher serum concentrations of sICAM-1 (p<0.001), sVCAM-1 (p<0.001), sE-selectin (p<0.01), and VEGF (p<0.001) were detected in patients with RA. Raised concentrations of sICAM-1, sVCAM-1, and VEGF dominated in the serum of patients with RA with follicular synovitis compared with those with diffuse synovitis (p<0.01 for all comparisons). The serum concentrations of sICAM-1, sVCAM-1, and VEGF correlated with markers of disease activity such as the erythrocyte sedimentation rate and C reactive protein levels. Furthermore, the clinical data analysed in our study indicated that patients with RA with follicular synovitis tend to have more severe disease. CONCLUSIONS: The distinct histological appearances of rheumatoid synovitis associated with different serum profiles of sICAM-1, sVCAM-1, and VEGF reflect varied clinical activity of the disease and confirm RA heterogeneity. Patients with different histological forms of synovitis may respond differently to the treatment regimens.

Serum matrix metalloproteinases and tissue inhibitors of metalloproteinases in different histological variants of rheumatoid synovitis.

OBJECTIVE: Rheumatoid synovitis is characterized by an invasive and tissue-destructive infiltrate of lymphocytes, macrophages and synoviocytes. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by these cells are important in the remodelling of the articular tissues in rheumatoid arthritis (RA). The aim of this study was to explore whether the serum concentrations of MMPs and their inhibitors were correlated with the histological appearance of the disease. METHODS: Tissue and serum samples were obtained from 37 patients with clinically active RA and 30 with osteoarthritis (OA). Morphological analysis allowed the division of RA synovial specimens into two distinct types. In 22 samples only diffuse infiltrates of mononuclear cells without further microanatomical organization were found. In 15 specimens we observed lymphocytic conglomerates with germinal centre-like structures. Serum concentrations of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase B (MMP-9), TIMP-1 and TIMP-2 were measured with an ELISA technique. RESULTS: Unique serum profiles of MMPs and TIMPs were identified in each of the two histological types of RA synovitis. The serum concentrations of MMP-1, MMP-3 and MMP-9 were higher in RA patients than in OA patients used as a control group (P<0.001 for all comparisons). These three MMPs dominated in the serum of RA patients with follicular synovitis compared with those with diffuse synovitis (P<0.05, P<0.01 and P<0.001 respectively). The analysis of the serum concentrations of TIMP-1 and TIMP-2 showed that their levels were also elevated in RA patients compared with OA patients (P<0.001 and P<0.01 respectively). Only TIMP-1 was found in a significantly higher concentration in the serum of RA patients with follicular synovitis than in those with diffuse synovitis (P<0.05). The serum concentrations of MMPs and TIMP-1 clearly identified patients with two different histological types of rheumatoid synovitis and with OA. Additionally, the analysis of clinical data showed that the rheumatoid disease in patients with follicular synovitis seemed to be more active than in those with diffuse synovitis. CONCLUSION: The morphological appearance of rheumatoid synovitis and the serum MMP and TIMP-1 profile were correlated with the clinical activity of the disease, confirming the heterogeneity of RA. These associations also suggest that patients with different histological forms of RA might require different treatment regimens.

Serum cytokines in different histological variants of rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by an invasive and tissue destructive infiltrate of lymphocytes, macrophages, and synoviocytes formed in the joints. Its etiopathogenesis and the role of the particular morphological components of synovitis remain unclear. There is evidence that its histological heterogeneity is correlated with synovium cytokine transcription. We investigated whether the serum cytokine profile is associated with the morphological appearance of the disease. METHODS: Tissue and serum samples were collected from 25 patients with clinically active RA and 25 with osteoarthritis (OA) as a control group. After histological analysis RA synovial biopsies were divided into 2 distinct types; 16 samples were characterized by diffuse lymphocyte infiltrates with no additional microanatomical organization. Lymphocytic aggregates with germinal center-like structures were found in 9 specimens. Serum concentrations of interferon-gamma (IFN-gamma), interleukin 12 (IL-12, p70 heterodimer), tumor necrosis factor-alpha (TNF-alpha), and IL-15 were measured by ELISA. RESULTS: Low concentrations of IFN-gamma (p < 0.01) and IL-12 (NS) were found in RA patients' serum compared with OA controls. RA patients with follicular synovitis had lower serum concentration of IFN-gamma (p < 0.05) and IL-12 (p < 0.05) than patients with diffuse infiltrates. High concentration of TNF-alpha and IL-15 characterized RA patient serum in comparison with controls (respectively, p < 0.001 and p < 0.01). In the serum of RA patients with follicular synovitis TNF-alpha was a dominant cytokine (p < 0.01) compared to patients with diffuse disease. At TNF-alpha level > or = 44 pg/ml, 5 (56%) of 9 patients with follicular RA had such elevated values vs one of 16 diffuse patients (< 10%; p < 0.02). Only serum concentrations of TNF-alpha could effectively differentiate between patients with OA and subgroups of RA. Analysis of clinical data suggested that activity of rheumatoid disease in patients with follicular synovitis was more severe than in those with diffuse infiltrates. CONCLUSION: The association between distinct histological appearance of rheumatoid synovitis and serum cytokine profile and diverse clinical activity of disease seems to confirm its heterogeneity.

Morphometric and ultrastructural studies of the sciatic nerve regeneration in rats intoxicated with ethanol.

The aim of the study was to examine the process of sciatic nerve regeneration and changes in the dorsal root ganglia (from which sensory fibres of the sciatic nerve extend) in animals intoxicated with ethanol. The experiment used 20 rats, divided into two groups: control and treated. The treated animals were intragastrically given 2g/kg b.w. of ethanol in 25% aqueous solution. In both groups the right sciatic nerve was transected and then sutured. After 5 months the animals were anaesthetized. The left and the right spinal dorsal ganglia-L5 and sections from the non-operated and operated sciatic nerves were collected for analysis. Ultrastructural examinations and morphometric measurements were conducted. It was found that ethanol administrated to rats inhibited regeneration of the transected and then sutured sciatic nerve, impairing the growth of axons in the transected nerve and destroying the regenerating sensory ganglion cells. The mechanism of the changes described may be associated with axonal transport disorders or with the suppressed production of biologically active substances, which affect nerve regeneration.

[Proliferative activity of glial neoplasms of the brain]. / Badania aktywnosci proliferacyjnej glejowych nowotworów mózgu.

The aim of the study including 89 brain gliomas was to determine their proliferative activity assayed with immunohistochemical methods (PCNA and Ki-67) and with the method of AgNORs, as well as to evaluate the correlation between the proliferative activity and features of histological malignancy. The study reveals that the estimation of PCNA, Ki-67 and AgNORs are effective methods for the determination of the proliferative activity of brain gliomas. Statistically significant differences were noted in the proliferative PCNA, Ki-67 and AgNORs between groups of gliomas with lower and higher malignancy, which indicated a distinct correlation between histological malignancy of the tumours and their proliferative activity. High values of PCNA and Ki-67 (> 40%) and AgNORs (> 15) were found to considerably deteriorate prognosis in brain gliomas.

[Expression of p53 protein and proliferative activity in multiform glioblastoma]. / Ekspresja bialka p53 i aktywnosc proliferacyjna w niedojrzalych glejakach wielopostaciowych.

The aim of the study of 41 multiform glioblastomas was the analysis of p53-protein immunoreactivity in neoplastic cells and evaluation of relationship of this biologic marker to tumour proliferation activity. Positive p53 expression was observed in 24 (58.5%) tumours, the negative one in 17 tumours (41.5%). Proliferation indexes of PCNA, anti-Ki6 and AgNORs showed high values in the multiform glioblastoma p53 positive group, but without statistical differences in comparison with the group of p53-negative glioblastomas. Significant differences were observed in survival time of patients with p53 positive tumours in comparison with p53-negative ones. In 15 patients with p53-positive multiform glioblastomas survival time was less than 6 months (62.5%) on the contrary with only 4 patients with similar survival time in p53-negative glioblastoma group (23.5%). Our results suggest that p53 expression in multiform glioblastoma cells, generally considered as the indirect index of p53 suppressor gene, reflects aggressive stadium of neoplastic disease and significantly worsens the prognosis.

AgNORs in duct epithelial lesions in chronic pancreatitis and in pancreas cancer cells.

BACKGROUND/AIMS: Argyrophilic nucleolar organizer regions (AgNORs) reflect the proliferative activity of cells. Since the majority of pancreatic cancers are ductal carcinomas, the aim of the study was to determine the AgNORs expression of potential pre-neoplastic ductal epithelial lesions in advanced chronic pancreatitis compared with pancreatic cancer cells. METHODOLOGY: Histological preparations obtained from 24 patients with chronic pancreatitis and 16 patients with pancreatic cancer were used to estimate the number of AgNORs per nucleus. Four types of AgNORs were distinguished and histograms with cell percentage of each type were performed for all forms of epithelial anomalies. RESULTS: In simple hyperplasia, squamous and mucous metaplasia the number of AgNORs ranged from 1.92 to 2.23; type I was predominant. In papillary hyperplasia, dysplasia and in situ carcinoma the number ranged from 2.98 to 3.34, with a predominance of type II-IV. In invasive carcinoma the number was 4.29 and 74% of cells were of type II-IV. CONCLUSIONS: Both counts of AgNORs and the percentage of type II-IV cells showed a gradual increase from simple hyperplasia through papillary hyperplasia and dysplasia to invasive carcinoma which in this respect differs significantly from all forms of the epithelial anomalies examined.

Epithelial anomalies in chronic pancreatitis as a risk factor of pancreatic cancer.

BACKGROUND/AIMS: The relationship between chronic pancreatitis and the development of pancreatic cancer is still a matter of dispute. Our aim was to determine the frequency of hyperplastic, metaplastic and dysplastic epithelial anomalies in the course of chronic pancreatitis and the potential steps in their development to malignancy. METHODOLOGY: The study was based on biopsy material of 70 patients with clinically diagnosed advanced chronic pancreatitis, who underwent partial or total pancreatectomy, as well as other operations. The patients were assigned to 2 groups: Group I (n = 41) with calcifying chronic pancreatitis; Group II (n = 29) with other forms of the disease. Histological sections were stained with hematoxylin-eosin, Mallory-azan, Gomori's silver method, and glycosaminoglycans (PAS and Alcian blue staining). Special interest was focused on the type and incidence of epithelial ductal and acinar cell anomalies, and on the degree of parenchymal scarring. RESULTS: Hyperplasia of the ductal epithelium was present in 31.4%, focal squamous metaplasia in 21.4%, mucous metaplasia in 11.1%, cellular dysplasia in 8.6%, dysplastic acinar cell nodules in 21.4%, and "tubular complexes" in 30.0% of all cases. The differences in the frequency of these changes, except for ductal epithelial hyperplasia, were not statistically significant in two comparable groups. Advanced pancreatic fibrosis was associated with epithelial anomalies in 65.7% of all cases. CONCLUSIONS: From the morphological point of view, the adequate prerequisites for the consideration of advanced forms of chronic pancreatitis, independent of type, as a risk factor of pancreatic cancer exist, necessitating the surgical removal of pathological lesions.

[Clinical significance of arthroscopic synovial biopsy in the diagnosis of knee synovitis]. / Przydatnosc kliniczna artroskopowej biopsji blony maziowej w diagnostyce wysiekowych zapalen stawów kolanowych.

Eighty-two patients (49 females and 33 males, mean age 38.2 years) underwent arthroscopic synovial biopsy in the course of treatment for chronic knee synovitis. After histopatological analysis of the samples Fritz et al. criteria were used for their classification. In 16 cases of specific synovitis clinical diagnosis was always confirmed by histopathology. In 66 patients with nonspecific synovitis lymphoplasmatic and serofibrous type prevailed. In 8 cases with dominant non-specific synovitis histological features of specific synovitis have been also found and correct clinical diagnosis has been established. Arthroscopic synovial biopsy increases diagnostic potential in the synovitis of unclear etiology.

Ultrastructural evaluation of microcirculatory vessels in rheumatoid synovial membrane.

Since morphological lesions in microcirculatory vessels are often difficult to be found in the light microscope, the electron microscope investigations were performed on the synovial membrane biopsy specimens from 70 patients with rheumatoid arthritis (RA). Most common lesions referred to venules, capillaries and arterioles were swelling and proliferation of endothelial cells, adherence of lymphocytes, monocytes and neutrofiles to the endothelium, their margination and diapedesis. Also destructive changes in the endothelial cells, basement membrane thickening due to their multiplication and microthrombi were observed. Platelet aggregates, fibrin, fragments of desintegrated cells and deposits of granulofibrillary substance corresponding to fibrinoid necrosis were frequently seen. In 7 patients, lesions of this kind were found only in electronograms. It can be assumed that the evaluation of ultrastructural changes in the microcirculatory vessels may be of great significance as a complementary diagnostic examination in future determination of RA progression and further prognostication.

C-reactive protein concentration in the sera of pregnant women with imminent premature parturition and preterm amniorrhea.

C-reactive protein (CRP) levels were evaluated during spontaneous labour, in imminent premature parturition (IPP) and after preterm rupture of oocyst membranes (PROM). Increasing CRP level during spontaneous labour was found in 16.6% parturients. Elevated CRP level in patients with IPP makes it impossible to predict premature labour. In parturients with PROM, increase in CRP level makes further infection predictable only in a few cases.

[Diagnosis usefulness of beta-2 microglobulin in clinical practice]. / Przydatnosc diagnostyczna beta 2-mikroglobuliny w praktyce klinicznej.

The laboratory diagnostics is mostly based on proteins determination and this tendency has been developed lately. It especially concerns the determination of the low molecular weight proteins. One of them became beta 2-microglobulin (beta 2-M), whose role in the organism and diagnostics is presently investigated. In this paper, the general characteristic of beta 2-M has been given including the usefulness in clinical diagnostics. A great part of this paper concerns role of this protein in nephrological diagnostics (early detection and localization of disorder, selective evaluation of filtration and resorption function, assessment of the degree of glomerules and tubules damage) and its significance in this field is without question. A very important problem is the determination of beta 2-M both in serum and urine, giving full information about renal dysfunction. The usefulness of beta 2-microglobulin in diagnosing cancer also has been described and discussed, and this protein can be used as a relatively sensitive marker of treatment in selected leukaemias. A beta 2-M usefulness in diagnostics has been presented in other diseases too, but it is worthy to note that the value of this test presently is not fully appreciated by clinicians.