Comparison of P19-derived neuroprogenitor and naive cell survival after intracerebellar application into B6CBA mice.

Mouse embryonic carcinoma cells (P19 line) were studied for both their survival and developmental potential in the intact cerebellum of B6CBA mice. The P19 cells were cultured and labelled with green fluorescent protein using transfection. Cells were used for transplantation either in the undifferentiated stage or after 3 days of neurodifferentiation induced by retinoic acid. The intracerebellar application was performed in 43 mice: group A (N = 21) received neuroprogenitors and group B (N = 22) received undifferentiated cells. The morphology of transplanted cells within the context of the surrounding cerebellar tissue was evaluated after 3 weeks. Naive P19 cells engrafted and survived in the cerebellum of 7 of the 22 adult mice (survival rate 31.8 %). Neuroprogenitors survived in 13 of the 21 mice (survival rate was 61.9 %). Since the cut-off is P < 0.05, the difference is not statistically significant (P = 0.069). An expansive appearance of the graft was significantly more frequent (P = 0.0047) in naive P19 cells than in neuroprogenitors. In mice in which the grafts did not survive, no marks of grafted cells or only fluorescing detritus were found. In conclusion, this is the first study to track the fate and morphology of embryonic carcinoma cells transplanted into the cerebellum, confirming that neuroprogenitors derived from embryonic carcinoma cells can settle in the host tissue and differentiate according to the surrounding conditions. With further validation, the embryonic carcinoma cells could become a valuable model with which to study the impact of cell therapy on neurodegenerative diseases.

Characterization of P19 cells during retinoic acid induced differentiation.

The aim of our study was to characterize mouse embryonal carcinoma (EC) cells P19 in different stages of retinoic acid induced neurodifferentiation by two methods, immunocytochemistry and RT qPCR. The characterization of the cells is crucial before any transplantation into any model, e.g. in our case into the mouse brain with the aim to treat a neurodegenerative disease. Specific protein markers (MAP-2, OCT-4, FORSE-1) were detected by immunocytochemistry in the cell cultures. The mRNA expression levels of PAX-6, MASH-1, Brachyury, GATA-4 and AFP were determined by RT qPCR method. HPRT was used as a housekeeping gene. The degree of differentiation can be characterized by expression of analyzed genes. The presence of OCT-4 and FORSE-1 proteins in undifferentiated pluripotent cells and the presence of dendrite specific MAP-2 in neuroprogenitors was detected. The expression levels of PAX-6 and MASH-1 increased and expression of Brachyury decreased during the neurodifferentiation process. The expression levels of GATA-4 and AFP were the highest after induction of differentiation with retinoic acid. Detailed characterization of cells before transplantation experiments can contribute to better understanding of their effect.

Leukaemia inhibitory factor (LIF) gene mutations in women diagnosed with unexplained infertility and endometriosis have a negative impact on the IVF outcome. A pilot study.

The frequency of functionally relevant mutations of the leukaemia inhibitory factor (LIF) gene in infertile women is significantly enhanced in comparison with fertile controls. The objective of this retrospective cohort study was to evaluate the impact of LIF gene mutations on the outcome of the treatment in women with various causes of infertility. Fifteen infertile women with the G to A transition at position 3400 leading to the valine to methionine exchange at codon 64 were analysed. Group A was made up of women with diagnoses that are frequently accompanied by changes in humoral as well as cell-mediated immunity - idiopathic infertility and endometriosis (N = 7). Group B consisted of patients with polycystic ovary syndrome (PCOS), andrological factor, tubal factor and hyperprolactinaemia (N = 8). The control group comprised 136 infertile women with no LIF gene mutation diagnosed with idiopathic infertility and endometriosis (N = 37) (group C) and patients with PCOS, tubal and andrological factor (N = 99) (group D). Seven of the mutation-positive patients were successfully treated by in vitro fertilization (IVF), but nobody in this group was diagnosed with idiopathic infertility and only one with endometriosis, which means that there is a statistically significant difference in the pregnancy rates between groups A and B (P = 0.01, Fisher's 2 by 2 exact test) but no statistically significant difference when comparing patients with the LIF gene mutation (group A+B) to no LIF gene mutation (group C+D). The results suggest that in mutation-positive women the idiopathic infertility and endometriosis have a negative impact on the outcome of IVF treatment.

Kissing stents in the aortic bifurcation--a valid reconstruction for aorto-iliac occlusive disease.

OBJECTIVE: To evaluate outcome and patency predicting factors of kissingstent treatment for aorto iliac occlusive disease (AIOD). METHODS: Patients treated with kissingstents for AOID between 1995 and 2004 at a tertiary referral center were identified through local databases. Chart review and preoperative images were used for TASC and Fontaine classification. Follow-up consisted of clinical exams, ABI and/or duplex. Patency rates were estimated by Kaplan-Meier analysis, and Cox multivariate regression was used to determine factors associated with patency. RESULTS: 173 consecutive patients (46% male, mean 64 years) were identified. TASC distribution was: A 15%, B 34%, C 10%, D 41%. Mean follow-up was 36 months (range: 1-144). 30-day mortality was 1% (2 patients), and 1-year survival was 91% (157 patients). 2 patients underwent late, open conversion and 13 patients suffered minor puncture site complications. Primary, assisted primary and secondary patency was: 97%, 99% and 100%, and 83%, 90% and 95% at twelve and 36 months respectively. There was no significant difference in patency between the TASC groups. Patency was significantly worse for patients in Fontaine class III. CONCLUSIONS: Aortoiliac kissing stents is a valid alternative to open repair for TASC A-D lesions. The procedure has low mortality and morbidity and good patency at 3 years.

[How can the haematopoietic stem cells from the umbilical cord blood be de-differentiated in vitro? Our first results using the co-cultivation systems]. / Jak dediferencovat hematopoetické kmenové bunky z umbilikální krve in vitro? Nase první výsledky s pouzitím kokultivacních systému.

OBJECTIVE: Aim of this study was to de-differentiate the haematopoietic stem cells (HSCs) that originated from the umbilical cord blood. One of the ways to do it is to use a co-cultivation system. DESIGN: Prospective experimental study. SETTING: Laboratory study - Institute of reproductive medicine and endocrinology, Pilsen. METHODS: HSCs were co-cultivated with mouse embryonic stem cells (mESC) with and without feeder cells. After co-cultivation HSCs were analyzed using flow-cytometry for presence of haematopoietic markers (CD34, CD45, CD133) and using immunohistochemistry for presence of embryonic stem cell markers (SSEA-4, Tra-1-60, Tra-1-81). RESULTS: No de-differentiation was detectable in any our experiment, only the intensity of the HSC cell markers decreased. CONCLUSION: We suppose that there were two major reasons for the experiment failure: there was no direct cell to cell contact and there was a mixture of cell types that originated from two different species. To reach our goal of in vitro de-differentiation we will need to change our strategy towards a pure human culture system without any animal additives and with cell to cell contact.

[The leukemia inhibitory factor gene mutations in the population of infertile women: the heterozygote transition G to A on the position 3400 does not affect the outcome of the infertility treatment]. / Mutace v genu pro leukemický inhibicní faktor v populaci neplodných zen: heterozygotní bodová zámena G za A na pozici 3400 neovlivnuje úspesnost lécby.

OBJECTIVE: The leukemia inhibitory factor (LIF) is one of the most important signaling factors in the embryo-maternal cross talk during the embryo implantation. We investigated the prevalence of the LIF gene mutations in the population of infertile women and their impact on infertility treatment. DESIGN: A cohort study. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine and University Hospital of Charles University, Pilsen. SUBJECTS AND METHODS: The population to screen consisted of 399 infertile women. The control population was comprised of 202 healthy fertile subjects. For the mutational analysis, the temperature gradient gel electrophoresis (TGGE) followed by subsequent sequencing of the positive samples, had been used. The groups of fertile controls and infertile patients were compared for statistically significant difference using the Fisher's 2 by 2 Exact test. RESULTS: Twelve potentially functional LIF gene mutations, the G to A transversion at the position 3400 leading to the valin to methionin exchange at codon 64 (V64M) were detected in the group of infertile women. No mutations were identified in the control group, which means that the frequency of functionally relevant mutations of the LIF gene in infertile women is significantly enhanced in comparison with controls (P = 0.01, Fisher's 2 by 2 Exact test ). Seven of these patients were successfully treated by in vitro fertilization (IVF). CONCLUSION: The results suggest that the LIF gene mutation, the heterozygote G to A transition on the position 3400, affects fertility but the infertility treatment can succeed. Even though LIF gene mutations occur infrequently and can be overcome by infertility treatment, their impact on molecular events during early phases of pregnancy should be further elucidated.

Association of the leukemia inhibitory factor gene mutation and the antiphospholipid antibodies in the peripheral blood of infertile women.

To characterize the impact of the potentially functional mutation--the G to A transition at the position 3400 of the leukemia inhibitory factor (LIF; a pluripotent cytokine that plays a central role in the control of the embryo implantation) gene that leads to the exchange of valine with methionine at codon 64 we evaluated the association of the LIF gene mutation and the levels of antiphospolipid antibodies (aPLs) in the peripheral blood of infertile women (the aPLs examination was part of our routine immunological test during the infertility check-up). Eight infertile mutation-positive women were diagnosed with idiopathic infertility (n=5) and endometriosis (n=3) and their levels of aPLs in serum were compared with 115 infertile women without any LIF gene mutation. Enzyme-linked immunosorbent assay was used for the detection of seven antiphospholipid antibodies; the results were statistically assessed by the Fisher's 2 by 2 exact test to evaluate the association of the LIF gene mutations and aPLs in serum of infertile patients. The presence of aPLs was significantly higher in our study group (100%) than in 30% of aPLs-positives in control infertile patients (p = 0.0035) which indicates that the aPLs are elevated in women with LIF gene mutations.

Endovascular recanalisation in occlusive mesenteric ischemia--feasibility and early results.

OBJECTIVE: To evaluate a single centre experience of endovascular treatment of mesenteric ischemia caused by vascular occlusion. DESIGN: Retrospective study. MATERIAL AND METHODS: Between 1995 and 2002 17 patients (12 females; mean age 61 years) with symptoms of bowel ischemia were treated endovascularly for arterial occlusion. Vessels were evaluated with angiography and pressure gradient measured. A mean gradient of > 20 mmHg or a stenosis of > 50% was considered significant. Patient data were recorded prospectively and follow-up was supplemented with retrospective chart review. Fifteen patients had follow up imaging, median 10 months (3-29 months) postoperatively. Median clinical follow up was 14 months (5-42 months). RESULTS: Recanalisation was successful in 16 patients (94%). The average number of stents used was 1.6 per patient. For one patient recanalisation failed with subsequent SMA dissection. A celiac artery stenosis was stented but symptoms remained postoperatively. Perioperative mortality was 5.8% (n = 1). 14/17 patients (82%) displayed symptom relief/improvement. Six patients required secondary endovascular intervention; PTA (n = 3); stent/stentgraft (n = 3). Two of these patients required a third procedure. 4/6 patients that underwent secondary intervention were asymptomatic and of recurrent stenosis > 75% and a gradient > 15 mmHg mean pressure gradient on imaging. Two patients were treated because of a combination of angiographic findings and/or significant pressure gradient combined with clinical symptoms. CONCLUSIONS: Endovascular treatment of mesenteric ischemia due to vessel occlusion is feasible with acceptable short-term results and limited complications. Most patients experience relief/improvement of symptoms. A significant number of patients might need endovascular re-intervention because of restenosis.

Stent-graft repair of a splenic artery aneurysm.

We present a case of splenic artery aneurysm (SAA) treated with stent-grafts. This new method offers the benefit of preserving the blood flow through the splenic artery. This in turn allows for subsequent sequential embolization of the spleen when indicated, as in our patient with hypersplenism. This is the first reported case of stent-graft repair of SAA.

Primary stenting in infrarenal aortic occlusive disease.

PURPOSE: To evaluate the results of primary stenting in aortic occlusive disease. METHODS: Thirty patients underwent primary stenting of focal concentric (n = 2) and complex aortic stenoses (n = 19), and aortic or aorto-iliac occlusions (n = 9). Sixteen patients underwent endovascular outflow procedures, three of whom also had distal open surgical reconstructions. Median follow-up was 16 months (range 1-60 months). RESULTS: Guidewire crossing of two aorto-biiliac occlusions failed, resulting in a 93% (28/30) technical success. Major complications included one access hematoma, one myocardial infarction, one death (recurrent thromboembolism) in a patient with widespread malignancy, and one fatal hemorrhage during thrombolysis of distal emboli from a recanalized occluded iliac artery. One patient did not improve his symptoms, resulting in a 1-month clinical success of 83% (25/30). Following restenting the 26 stented survivors changed their clinical limb status to +3 (n = 17) and +2 (n = 9). During follow-up one symptomatic aortic restenosis occurred and was successfully restented. CONCLUSIONS: Primary stenting of complex aortic stenoses and short occlusions is an attractive alternative to conventional surgery. Larger studies with longer follow-up and stratification of lesion morphology are warranted to define its role relative to balloon angioplasty. Stenting of aorto-biiliac occlusions is feasible but its role relative to bypass grafting remains to be defined.

Percutaneous placement of stents in chronic iliac and aortic occlusive disease.

OBJECTIVES: to evaluate the long-term results of stenting in iliac and aortic occlusive disease and identify factors predicting clinical outcome. DESIGN: retrospective study. MATERIALS AND METHODS: 82 patients (87 limbs) with stenoses (n =63) and occlusions (n =24) in the aortoiliac segment were treated with stents. 81 lesions involved the iliac arteries, 3 the infrarenal aorta and 3 the aortic bifurcation. Primary stenting was performed in 57 limbs (complex stenotic lesions and occlusions). Stents were placed following failed PTA in 30 limbs. Median follow-up was 13.2 months (1-66 months). RESULTS: technical success was 99% and clinical success 89%. Cumulative primary and secondary patency of stented iliac atherosclerotic lesions at 1 and 3 years was 75 and 61%, and 83 and 75% respectively. The factors predicting outcome of primary patency identified in Cox multivariate analysis were ankle-brachial index (ABI) prior stenting (p =0.03) and length of the lesion (p =0. 007). Major non-fatal complications occurred in 7.4% of the patients and there were no deaths attributed to the treatment. The 30-day mortality was 3.7%. CONCLUSION: stenting of complex aortoiliac stenoses and chronic occlusions is a safe and effective treatment modality. Long lesions and lower pre-procedure ABIs were found to negatively influence outcome.

Chronic aortic dissection: stenting of aortic true lumen obliteration with late dynamic variations of both lumens.

Percutaneous endovascular techniques were used to treat an arteriovenous fistula (AVF) associated with pancreatic transplantation. A pancreatic transplant superior mesenteric artery-to-superior mesenteric-vein AVF was successfully embolized while flow to the pancreas transplant was preserved. The embolization was aided by the use of Guglielmi detachable coils and a detachable balloon. No complications were encountered. At 23 months follow-up, the patient is doing well with no recurrence.

[Chronic mesenteric ischemia. Endovascular treatment is as effective as open surgery]. / Kronisk mesenteriell ischemi. Endovaskulär behandling lika effektiv som öppen kirurgi.

Chronic mesenteric ischaemia is a rare but serious condition, which if untreated may cause death secondary to starvation or bowel infarction. As the symptoms are sometimes unspecific, its diagnosis may be delayed or missed. Although open surgical revascularisation has been the traditional treatment, a review of published reports suggests it to be associated with operative mortality rates of 6-9 per cent, and major morbidity rates of 22-26 per cent. Reports by others, and our own experience, suggest that endovascular treatment of mesenteric atherosclerotic obstructions with PTA (percutaneous transluminal angioplasty) and stenting may yield patency rates differing little from those associated with surgery, but significantly lower mortality (1.6%) and morbidity (5.6%).

Endovascular treatment of chronic mesenteric ischemia: report of five cases.

PURPOSE: To evaluate the midterm results of percutaneous transluminal angioplasty (PTA) and stent placement in stenotic and occluded mesentesic arteries in five consecutive patients with chronic mesenteric ischemia. METHODS: Five patients with 70%-100% obliterations of all mesenteric vessels resulting in chronic mesenteric ischemia (n = 4) and as a prophylactic measure prior to abdominal aortic aneurysm repair (n = 1) underwent PTA of celiac and/or superior mesenteric artery (SMA) stenoses (n = 2), primary stenting of ostial celiac occlusions (n = 2), and secondary stenting of a SMA occlusion (n = 1; recoil after initial PTA). All patients underwent duplex ultrasonography (US) (n = 3) and/or angiography (n = 5) during a median follow-up of 21 months (range 8-42 months). RESULTS: Clinical success was obtained in all five patients. Asymptomatic significant late restenoses (n = 3) were successfully treated with repeat PTA (n = 2) and stenting of an SMA occlusion (n = 1; celiac stent restenosis). Recurrent pain in one patient was interpreted as secondary to postsurgical abdominal adhesions. Two puncture-site complications occurred requiring local surgical treatment. CONCLUSIONS: Endovascular techniques may be attempted prior to surgery in cases of stenotic or short occlusive lesions in patients with chronic mesenteric ischemia. Surgery may still be preferred in patients with long occlusions and a low operative risk.

Aggressive treatment of acute limb ischemia due to thrombosed popliteal aneurysms.

OBJECTIVE: The absence of infra-popliteal runoff in patients with acute limb ischemia and thrombosed popliteal aneurysms carries a high risk of amputation. A combined treatment method involving thrombolysis and surgery is reported. MATERIAL AND METHODS: Information regarding six patients was reviewed. Ankle brachial indices and degree of ischemia were recorded. All patients underwent digital subtraction angiography. In five patients thrombus dissolution was achieved using a combination of mechanical and pharmacologic therapy. One patient was judged incapable of withstanding any delay in reperfusion and was treated with isolated limb perfusion using a thrombolytic agent. All patients underwent surgical revascularization. Follow-up (1-3 years) consisted of duplex examinations at 6 months and yearly thereafter. RESULTS: Five patients had no measurable ankle brachial index (ABI), while one patient had an ABI of 0.4. Initial angiography noted all patients to have no runoff in continuity to the pedal arch. Following thrombolytic therapy, an adequate bypass vessel was noted in all cases, with reconstitution of the plantar arch in five patients. Distal revascularizations included one peroneal, and five below knee popliteal arterial bypasses. Fasciotomies were performed in four of the six patients. There were no amputations. One patient developed a persistent foot drop. Two patients developed bypass grafts occlusions; one of which required therapy. CONCLUSION: The pre-operative use of thrombolytic therapy is a safe and effective method to achieve limb salvage in this patient population. Patients must be capable of withstanding an additional period of ischemia allowing for reconstitution of distal runoff. Isolated limb perfusion is of use when a delay to reperfusion cannot be tolerated.

Aneurysms of the pancreaticoduodenal artery associated with occlusion of the celiac artery.

Aneurysms of the pancreaticoduodenal artery are rare and may be associated with celiac artery stenosis or occlusion. Twenty-eight patients are reported in the literature. The diagnostic findings and therapeutic alternatives of four additional patients form the basis of this report. One patient with ruptured pancreaticoduodenal aneurysm was successfully treated by transcatheter embolization, and one patient was treated surgically; both patients had an uneventful recovery. In the remaining two patients, the aneurysms were left untreated. One patient died 1 year later of an unrelated cause, and the other patient is symptom-free after 2 years.

Modes of action of colicins E4-E7: rates of basic biosyntheses inhibition.

The progress of the inhibition of prominent biopolymer syntheses (namely of DNA, RNA and protein synthesis) in cultures of susceptible bacteria Escherichia coli treated with colicins E4, E5, E6 or E7 was followed. The method used was the measurement of incorporation of specific radioactive precursors into an instantly precipitable fraction. All the three syntheses being inhibited successively, progressively and with different urgency during unfolding of the lethal effect of each colicin, emphasis was put on a comparative mathematical analysis of the development of the biosyntheses inhibition rates. In bacteria treated with colicins E4, E5 or E6, protein synthesis was blocked preferentially and most heavily, followed by cessation of DNA synthesis and finally also by a rather slowly proceeding impairment of RNA synthesis. (In E6-treated cells, damage to DNA synthesis started prior to that to protein synthesis.) Thus, the effect of colicins E4, E5 and E6 follows the general pattern of colicin E3 action. Bacteria treated with colicin E7 developed an immediate block of DNA synthesis, soon followed by protein and (again after a significant delay) by RNA synthesis switch-off. Thus the action of colicin E7 is closely related to that of colicin E2. The presumptive direct targets of these colicins remain to be elucidated.