[Comparison of growth of the follicle and mesenchymal stem cells to urothelial cells and fibroblasts on collagen scaffold]. / Porównanie wzrostu komórek macierzystych mieszków wlosowych i szpiku kostnego do wzrostu komórek urotelialnych i fibroblastów na matrycy z kolagenu typu I.

UNLABELLED: To develop a tissue-engineered bladder wall replacement with elements obtained from non-urinary tract components is an atractive idea. The aim of this study was to compare growth of hair follicles epithelial stem cells and mesenchymal stem cells to urothelial cells and fibroblasts cells on scaffold prepared from rat collagen type I. MATERIALS AND METHODS: Wistar rats were used in experiment. Rat urothelial cells, hair follicles epithelial stem cells, mesenchymal stem cells and 3T3 cells were cultivated in DMEM (Sigma) supplemented with 10% (or 20% for hair follicles cells) of Fetal Bovine Serum (FBS). Epithelial cell cultures were suplemented with EGF (10 ng/ml; Sigma). Cells were stained using anti-cytokeratine (Clone MMF) and anti-cytokeratine 7. Anti-CD34 and anti-p63 staining were done. Collagen scaffold was prepared from tendoms of Wistar rat's tails. 6-well plates were covered with collagen scaffold. 25 x 10(3) of cells were seeded on each well and cultured for a week. Cells in the controls were seeded on polystyrene surface. After a week cell viability was assessed using MTT test (Sigma). Each experiment was triplicated. Photo documentation was prepared. The differences between means were compared using t-Student test. RESULTS: There were 106.5 +/- 23.4 x 10(3) and 310.7 +/- 60.7 x 10(3) of 3T3 fibroblasts growing on polystyrene and collagen, respectively (p < 0.05). The initial cell number was 25.0 x 10(3). Urothelial cells expressed epithelial markers. There were 40.0 +/- 4.2 x 10(3) and 4.5 +/- 1.8 x 10(3) urothelial cells growing on polystyrene and collagen, respectively after 7 days of culture (p < 0.01). There were 118.5 +/- 19.7 x 10(3) and 114.1 +/- 33.2 x 0(3) of mesenchymal stem cells growing on polystyrene and collagen, respectively (NS). Hair follicles epithelial cells expressed epithelial markers and were slightly positive for CD34 and p63. There were 292.5 +/- 33.3 x 10(3) and 167.4 +/- 24.9 x 10(3) of hair follicles epithelial cells growing on polystyrene and collagen, respectively (p < 0.05). Collagen scaffold decreased proliferation of follicle epithelial and urothelial cells. CONCLUSIONS: Hair follicles epithelial stem cells and mesenchymal stem cells can be potentially used in tissue-engineering, with the guarantee of the sufficient cell number for transplantation. It seems that construction in vitro of urinary bladder walls from elements obtained from non-urinary tract tissues is feasible.

Autologous cultured melanocytes in vitiligo treatment.

BACKGROUND: Surgical treatment of vitiligo is indicated when lesions are localized in poorly responding areas. OBJECTIVES: The objectives were: (1) to establish the melanocyte culture obtained from the epidermis of vitiligo patients for future treatment; (2) to estimate the influence of selected factors on the formation of suction blisters and the results of culture; and (3) to compare the results of treatment of vitiliginous macules localized in the dorsum of the hands and lower limbs by transplantation of cultured autologous melanocytes plus psoralen and ultraviolet A (PUVA) therapy (CMP), suction blister transplantation plus PUVA therapy (SBP), cryotherapy plus PUVA-therapy (CP), and only PUVA therapy (OP). METHODS: Forty patients were qualified for the study. The roofs of the suction blisters were used as a melanocyte source for culture establishment or were directly transplanted. RESULTS: The CMP procedure was successfully performed on only 10 of 20 patients because of the difficulties in cell culture establishment. The SBP method was carried out on all 20 patients. A total lack of effectiveness was found in CP and OP methods. CONCLUSIONS: The effectiveness of culture depends on time of suction blister forming, phototype, and previous PUVA therapy. This study demonstrated the advantage of the SBP over the CMP method.

[Bone marrow isolated micrometastases of non-small cell lung cancer]. / Mikroprzerzuty niedrobnokomórkowego raka pluca do szpiku.

UNLABELLED: The ability to detect occult systemic metastases in patients with operable NSCLC could have a significant impact on the management of the disease. The aim of the study was to detect occult micrometastatic tumor cells in bone marrow in patients with resectable NSCLC. MATERIAL AND METHODS: A total of 35 patients (29 men, 6 women), age between 47 and 78 (mean 61.6) were included in the study. In each of the patients bone marrow aspirates from the ribs were sampled during surgery. Both the tumor and the bone marrow aspirate were examined histologically and immunocytochemically with the cytokeratin: AE1/AE3, CAM 5,2, CK-7, CK-18. The presence of grow factors CD 31 and CD 34 were examined as well. RESULTS: No evidence of micrometastases or tumor cells in bone marrow was found in histological examination. Cytokeratin positive (CAM 5,2 +) cells were detected in 33 cases (94.23%) of the tumors and in 21 cases (60.00%) of bone marrow samples. The statistically significant correlation between the presence of CAM 5,2 in tumors and bone marrow was found (p = 0.049). Cytokeratin positive cells were detected in all the 35 tumors (AE1/AE3), in 20 tumors--57.14% (CK-7) and in 23 tumors--65.71% (CK-18). Cytokeratin positive cells (CK-7) were detected in bone marrow sample in one patient only. CONCLUSIONS: Immunocytochemical examination with the use of cytokeratin CAM 5,2 is of use to detect occult micrometastatic tumor cells in bone marrow in NSCLC patients. However, no correlations were found between the presence of cytokeratin CAM 5,2 in bone marrow or tumor and patients' age, sex and the histological type of NSCLC its degree of malignancy and stage.

[Eyelid conjunctiva can be a potential cells source for conjunctival sack reconstruction purpose]. / Spojówka powiekowa moze byc potencjalnym zródlem komórek w chirurgii rekonstrukcyjnej worka spojówkowego.

UNLABELLED: There is no ideal culture establishing method for the conjunctival sac reconstruction. Two methods of primary conjunctival culture establishing were compared. MATERIALS AND METHODS: The internal surface of rat's eyelid was used. In the 1st method cells migrate from specimen and attached to the surface of culture plate. In the 2nd method tissue was digested and placed in the flask. Epithelial character of cultured cells was confirmed. RESULTS: Both methods allow to set up the culture of epithelial cells. Efficiency of enzymatic isolation was higher then growing from previously attached specimen. At the end of the experiment the cultures were similar. CONCLUSIONS: Both methods allow to establish the primary culture of conjunctival epithelial cells. Eyelid conjunctiva can be a cells source for the conjunctival sack reconstruction purpose.

Establishing melanocyte cultures in a serum-free system for transplantation in vitiligo patients.

BACKGROUND: The aim of the study was to establish melanocyte cultures obtained from the epidermis of vitiligo patients for future treatment and to estimate the influence of selected factors on the formation of suction blisters and the results of culture. MATERIAL/METHODS: Twenty patients with generalized vitiligo were qualified to the study. The melanocyte donor site was the arm or forearm where, using an electric vacuum suction machine and a plastic plate, suction blisters were formed. The removed roofs of the blisters were used as a melanocyte source for culture establishment. After an enzymatic bath, the cell suspension was transferred to culture flasks containing melanocyte growth medium (MGM M2). The melanocytes in culture were identified by immunocytochemistry. RESULTS: Suction blisters were formed in all patients. No melanocyte cultures were established from patients whose blisters appeared over a period 58 minutes. In addition, a suitable quantity of melanocytes was not obtained for eight other patients (with skin phototype II and/or without previous PUVA therapy). CONCLUSIONS: The epidermis of the arm and forearm is a good donor site for the establishment of melanocyte cultures. The effectiveness of culture depends mainly on the time of the formation of suction blisters, the phototype of the skin, and previous PUVA therapy.

The NOD2 3020insC Mutation in Women with Breast Cancer from the Bydgoszcz Region in Poland. First Results.

The frameshift NOD2 gene mutation 3020insC is predominantly associated with Crohn's disease, but predisposes to many types of common cancers as well. We studied the frequency of this mutant NOD2 allele in 148 breast cancer women from the Bydgoszcz region in Poland. The NOD2 mutation was present in 8.8% of the patients. The mean age at breast cancer diagnosis of the mutation carriers was 43 years. We did not find any mutation in patients diagnosed with breast cancer after the age of 50 years. There was no association of the NOD2 mutation with a strong family history of breast cancer. On the contrary, the mutation frequency (11.4%) was two times higher in women from families with a single case of breast cancer and with aggregation of other common types of cancer, especially digestive tract cancers. Low risk of breast cancer in the mutation carriers seems to be confirmed by finding the 3020insC mutation in three healthy parents of probands aged 73, 74 and 83 years, from three separate families.

[The role of preoperative hooked-wire localization of occult lesions for early detection of breast cancer]. / Rola przedoperacyjnego znakowania zmian niepalpacyjnych dla wczesnego wykrywania raka sutka.

OBJECTIVES: Nonpalpable small lesions, usually few millimeters in diameter, consist an important problem in the early diagnostics of breast cancer. DESIGN: An aim of this paper was an evaluation of usefulness of preoperative hooked-wire localization of such lesions for the detection of breast cancer. MATERIALS AND METHODS: Material consisted of 131 patients aged 37-77 in whom 132 procedures of preoperative hooked-wire localization were performed. The correlation between the mammographic findings and the cytologic or histologic results were then evaluated, with the histologic results being treated as standard. RESULTS: Histologic examination of the localized lesions revealed benign changes in 71.2%, atypical ductal hyperplasia in 4.6% and cancer in 24.2%. Positive predictive value for mammography was 24%. Significant correlation was seen between the suspicion of cancer in mammography and the histologic results. CONCLUSIONS: Preoperative localization should be used as a method of choice in radial lesions, suspected clustered microcalcifications and after the positive result from SFAB.

Abdominal wall repair using a biodegradable scaffold seeded with cells.

BACKGROUND/PURPOSE: The repair of large abdominal wall defects is still a challenge for pediatric surgeons. Synthetic materials, however, may lead to high complication rates. This study was aimed at applying tissue-engineering methods to abdominal wall repair. METHODS: 3T3 mouse fibroblasts were expanded in vitro. In the next step, a biodegradable material--polyglycolic acid (PGA)--was actively seeded with 10(7) cells/scm of PGA scaffold. Culture medium (Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum) was changed every 6 hours after seeding cells on PGA fibers. Under general anaesthesia, C57BL/6J black mice underwent creation of a 2 x 3-cm abdominal wall defect (60%-70% of abdominal surface). The defect was repaired in the experimental group with the fibroblast-seeded PGA scaffold. In the first control group, the defect was covered with acellular PGA, and in the second control group, by skin closure. Animals were killed after 30 days to assess the histologic and gross findings. RESULTS: No abdominal hernia was found in animals repaired with cell-seeded and acellular scaffolds. All animals with skin closure died within 7 days. In every case, tissue-engineered construct was thicker then in controls. Histologic and gross examination revealed a good neovascularisation in tissue-engineered abdominal walls comparing to the acellular matrix. There was no intensive scar formation between abdominal wall and skin. CONCLUSIONS: Engineered soft tissue constructs can provide structural replacement of severe and large abdominal wall defects. Tissue engineering in the near future will possibly enter clinical practice.

[Silicone and latex urinary catheters cytotoxicity on primary cultured rabbit urothelial cells]. / Cytotoksyczny wplyw silikonowych i lateksowych cewników urologicznych na pierwotna hodowle komórek nablonka pecherza moczowego królika.

UNLABELLED: In everyday urological practice, the use of urinary catheters creates problems such as epithelial hyperplasia, edema or erosion. THE AIM: of this study is estimation of the toxicity of latex and silicone urinary catheters on primary cultured rabbit urothelial cells (PRUCC) was compared. MATERIALS AND METHODS: Primary cultured rabbit urothelial cells (PRUC) were characterized for their proliferation, qualitative evaluation and quantitative measurement of cytokeratins. In the first kind of cytotoxic assay included exposition of the PRUC cells on liquid extracts of latex and silicone. Cytotoxicity of 1, 5, 10, 20, 50% dilution and pure extract was evaluated after 24 h. Additionally for 1% solution observation was extended up to 3 days. The second cytotoxic assay was based on the close contact between the catheter surface and cells. In direct contact test, latex and silicon catheters samples covered cultured cells. Trypan blue exclusion assay was used to measure cell viability. Cells were counted using inverted microscope with camera. RESULTS: 1, 5, 10 and 20% of silicon extracts did not affect cell viability while 5% of latex catheter extracts did. There was no difference in cytotoxicity when PRUC cells had been exposed to 1% dilutions of both silicone and latex catheters extracts after 3-days observation. 50% dilution and pure latex and silicone urinary catheters extracts appears to be highly toxic on PRUC. Direct contact test confirmed high toxicity of latex and silicone catheters on PRUC. CONCLUSIONS: We confirmed latex and silicone on PRUC to be very toxic with direct contact. Using extract test the properties of silicone seemed to be slightly better. Authors postulate using catheters covered with hydrogenic gel in every day urological practice.

[Fine-needle aspiration biopsy under CT-guidance--usefulness in diagnosis of malignant lung tumors]. / Znaczenie biopsji aspiracyjnej cienkoiglowej wykonywanej pod kontrola tomografii komputerowej w diagnostyce zlosliwych nowotworów pluca.

UNLABELLED: Aim of the study was to evaluate the usefulness of fine-needle aspiration biopsy (FNAB) in diagnosis of malignant lung tumors. MATERIAL AND METHODS: The results of 438 CT guided FNAB of lung lesions performed between Sep. 1st 1999 and Feb. 20th 2004 were evaluated. RESULTS: In 295 cases (67.35%) the cytological diagnosis of lung tumours were established by FNAB. The most common type of the tumours were: non-small cell lung cancer: 222 (50.69%) and small cell lung cancer: 49 (11.19%). 104 of these results were histologically verified and two false positive results were detected. In 143 cases (32.65%) however, the results established by FNAB were non-diagnostic. 63 of the lesions were verified and in 36 cases (57.1%) the malignant tumours were proved. The specificity of the method regarding malignant lesions reached 93.10%, and its sensitivity for patients with malignancy was 73.91%. The average diameter of the malignant tumour was 5.4+/-1.3 cm whereas in case of non-diagnostic results the diameter of the tumour was 4.1+/-1.6 cm. However, there was no significant difference between the two groups. The most common complication of FNAB was pneumothorax, which occurred in 22 patients (5.02%) and in 13 cases (2.97%) required drainage of the chest cavity. CONCLUSION: FNAB is a safe diagnostic method with high specificity and moderate sensitivity in detecting of malignant lung lesions.

Influence of autologous serum on rabbit chondrocytes proliferation in vitro. Comparing study to standard sera supplements.

Background. Autologous chondrocyte transplantation (ACT) is a method in reconstructive surgery. Fetal bovine serum (FBS) is still a "gold standard" for media supplementation in many laboratories. Prion and virus diseases are danger for patients treated witch ACT if FBS is used. The aim of this study was to establish the utility of media for chondrocytes culture supplemented with Autologous Serum (AS) or serum substitute (Ultroser G. UG) instead of FBS.
Material and methods. Primary rabbit articular chondrocytes culture was established and cultures in MHCH (Chondrocytes Culture Medium) medium with 10% FBS during first 3 passages. The influence of different sera in various concentrations of 10%, 5% and 2,5%, while Ultroser G in 2%, 1% and 0,5%. Ceels were transferred in amount of 104 into the 24-well plates containing tested media. After 96 hours cells were counted using trypan blue test and photo documentation was done. Means were compared using Student-T test.
Results. AS in concentration of 10% stimulated cell growth to 14,93+/-2,03 x 104 cell/well. Medium supplemented with the same concentration of FBS allowed to obtain 6,56+/-1,08 x 104 cell/well. Transfer from medium supplemented with 10% of FBS to flask containing Ultroser G stopped proliferation rate. Cell viability in AS and FBS supplemented media was over 90% while 80,8-8809% in UG.
Conclusion. 10% FBS and AS conditioned media are both suitable for chondrocytes culture. AS seems to be the serum conditioner for chondrocytes propagation.

[Coin lesion in the lung of patients with known malignancy]. / Cien okragly w polu plucnym u chorych na nowotwór zlosliwy.

Histological examination was performed on 93 pulmonary nodules resected in 90 patients with known malignancy. The diameters of the coin lesions on CT scan were 3-10 mm in 42 cases and 11-30 mm in 51 cases. The most common sources of the previous malignancy were: large bowel (20), breast (14), uterus (12), kidney (11) and larynx (7). In 84 cases the nodules were radically resected through "wedge" resection and in 9 cases lobectomy was performed. Most frequently, coin lesion was caused by metastases of known malignancy (in 69.9%), pulmonary fibrosis (in 11.8%) and by primary lung cancer (in 7.5%). In general, pulmonary nodules were found malignant in 78.5% and benign in 21.5% of the cases. In the subgroup of coin lesions of diameter between 11 and 30 mm malignancy was noted in 94.1%--significantly more frequently than in subgroup of diameter between 3 and 10 mm (59.5%). No significant correlation was found between histological type of pulmonary nodules (malignant or benign) and the type of primary malignancy as well as the time from the treatment of primary malignancy to the detection of the coin lesion.

Analysis of microsatellite instability and loss of heterozygosity in breast cancer with the use of a well characterized multiplex system.

Analysis of microsatellite instability (MI) and loss of heterozygosity (LOH) is recommended for screening patients with sporadic and hereditary malignancies. This study shows an application of a fluorescent hexaplex PCR system for microsatellite typing on A.L.F. DNA Sequencer (Pharmacia Biotech). This technique detects changes in microsatellites providing a time-efficient, reliable and accurate method for MI and LOH analyses. The Fragment Manager software was used for automated size calculation and quantitation of DNA fragments, enabling rapid and precise measurement of allelic ratios. We examined 70 breast cancer and 70 control DNA specimens, classified all the patterns of microsatellite alterations, and set up MI and LOH assessment criteria for the automated multiplex fluorescent method.