[Severe acute pancreatitis - managing complications using a combination of minimally invasive approaches]. / Tezká akutní pankreatitida - resení komplikací kombinací miniinvazivních prístupu

In this case report, we describe the case of a 30-year-old obese patient with severe acute pancreatitis complicated during hospitalization by the development of infected necrosis, a pseudocyst and an abscess. We demonstrate a possible solution to these complications using a combination of minimally invasive approaches. The contribution of this case report resides in that it presents a combination of the percutaneous approach and the endosonographic approach in the treatment of pancreatic necrosis. We believe that in such an extensive necrosis of the pancreas as is described in our case, this combined approach is optimal. The condition for implementing such a procedure is a well-staffed and technically equipped workplace.

In vitro evaluation of the permeation of cytotoxic drugs through reconstructed human epidermis and oral epithelium.

BACKGROUNDS: Occupational exposure to antineoplastic agents may represent a risk to health care workers, although the relevance of different exposure routes is not fully understood. The objectives of this study were to determine in vitro permeation of four widely used cytotoxic drugs (cisplatin, cyclophosphamide, doxorubicin, and fluorouracil) through two reconstituted tissue models representing human skin epidermis and oral mucosa. MATERIALS AND METHODS: Experiments were conducted with reconstructed models of human epidermis and oral epithelium, cultured in a chemically-defined medium under conditions simulating possible exposure scenarios (6 h duration, three concentrations corresponding to commonly used application doses). The amounts of drugs permeated through the tissues into the receptor media were determined using ultra performance liquid chromatography with photospectrometric detection. RESULTS: The highest epidermis permeations (P = 0.2 x 10(-3) - 1.5 x 10(-3) cm x h(-1)) were observed with three polar drugs (cisplatin, cyclophosphamide and fluorouracil), while permeation by more hydrophobic doxorubicin was minor (P(max) = 0.03 x 10(-3) cm x h(-1)). As expected, more pronounced tissue permeation was observed with the reconstructed oral epithelium having the maximum permeability coefficient (P = 180 x 10(-3) cm x h(-1)) for cisplatin and fluorouracil. Histological evaluation of the exposed tissues revealed cytotoxic effects at higher doses, especially for oral epithelium. CONCLUSION: Although the skin epidermis with keratinised stratum corneum provided relatively good protection, uptake (of at least some investigated drugs) via both types of tissue should not be underestimated. Our results provide basic experimental data on the skin and oral epithelia permeation for further modelling of exposure and health risk assessment.

[Evaporation of selected cytotoxic drugs and permeation of protective gloves--research into the occupational risks of health care personnel handling hazardous cytotoxic drugs (CYTO project)]. / Studie evaporace vybraných cytostatik a propustnosti ochranných rukavic v rámci výzkumu profesní záteze zdravotnických pracovníku exponovaných cytotoxickým protinádorovým lécivum (projekt CYTO).

BACKGROUNDS: The CYTO project studies an important aspect of healthcare provision -long-term occupational exposure, both threshold and below-threshold, to chemical agents with carcinogenic and mutagenic properties, with the major focus on antineoplastic drugs.This contribution presents experimental results from the first stages of the project's experimental work, i.e. an evaluation of the physico-chemical characteristics of cytostatic agents (evaporation) and an investigation into protective glove permeation. MATERIALS AND METHODS: In co-operation with IUTA (Institut für Energie- und Umwelttechnik e.V., Duisburg, Germany), the vapour pressure of paclitaxel, doxorubicin and dacarbazine was measured following OECD guideline No. 104: Vapour pressure curve--vapour pressure balance. Furthermore, the evaporation of cytostatic drugs was examined in actual laboratory conditions by monitoring the airborne concentration using the passive sampling technique. Besides the evaporation of selected drugs, the permeation of cisplatin, cyclophosphamide, doxorubicin, 5-fluorouracil and paclitaxel through different types of gloves (vinyl, latex, nitrile) was assessed. RESULTS: Although our experiments showed relatively slow evaporation of the evaluated cytostatic drugs (the highest pressure in paclitaxel was 0.024 Pa), equilibrium concentrations may go up to milligrams per cubic metre. Nevertheless, analytical measurements of airborne contamination did not confirm these concentration levels. The glove permeation experiments with cytostatics showed good resistance of nitrile gloves (which were impermeable to all five drugs). Other materials should be avoided while handling cytostatic agents (e.g. maximum permeation of cyclophosphamide through latex was 19 microg/sq cm/hr). CONCLUSION: Although the volatility of cytostatic agents is low, it cannot be neglected considering the chronic character of exposure. However, in order to estimate actual occupational exposure, future research should focus on the development of sensitive analytical methods. Nevertheless, dermal uptake is supposed to be the major route of exposure and use of protective gloves is necessary to minimize potential risks. Our simulated time-dependent permeation experiments with cytostatic agents and different glove materials showed that good protection is provided only by nitrile gloves. The results obtained in this study will be used for the modelling of exposure doses and health risk assessment in the subsequent stages of the CYTO research project.

[Differences in ulcer location in diabetic foot syndrome]. / Vliv etiologie syndromu diabetické nohy na lokalizaci defektu.

UNLABELLED: The foot ulcerations are among the most debilitating complications in diabetic patients. The main risk factors leading to the ulcer development are diabetic neuropathy (sensoric, autonomic), limb ischemia (angiopathy), limited joint mobility and teh plantar pressure; the infection plays a role in difficulty of ulcer healing. The aim of our study was to assess the possible differences in location of diabetic ulcers with regard to their origin. In 502 patients during 5 year interval 835 new diabetic ulcers were diagnosed. METHODS: Ulcers were divided into 3 groups according to their origin: neuropathic, neuroischemic and ischemic. RESULTS: In the neuropathic group most ulcers were found in the plantar surface of toes (40.4%) and in the plantar metatarsal heads region (39.1%); in contrast, the ischemic group had the most frequent location in the toe tips (63.6%), while the neuroischemic group had most ulcers distributed in both plantar surface and tips of the toes (51.8%). The ulcer distribution was statistically significant different in all groups and depended on the etiology of ulcers (p < 0.0001; Fisher's exact test, modification Monte Carlo). Totally more than 75% of all ulcer were located in the toe and forefoot area. The patients in the neuroischaemic group had more often revascularisation procedures. The patients in ischaemic group were more often after high amputation. These patients had always less microvascular diabetic complication (all p < 0.01; ANOVA chi2). CONCLUSION: The location of diabetic foot ulcers differs significantly according to their cause. In addition more than 75% of all ulcerations were localisated in toes and forefoot area. This fact could change focus of the preventive strategy in the diabetic foot.

The relationship between HA-1 compatibility and the activation of helper T lymphocyte precursors.

The relationship between the compatibility in minor histocompatibility HA-1 antigen and the activation of helper (IL-2 producing) T lymphocyte precursors in vitro was studied in the group of 17 HLA-A2 positive HLA identical siblings. Although the number of pairs studied is still small, no correlation has been found between HA-1 compatibility and helper T lymphocyte precursors activation. The results presented here could suggest the possibility that the HTLp assay does not have to be a relevant parameter for the detection of HA-1 mismatches in HLA identical siblings.

Transplantation of allogeneic peripheral blood progenitor cells--a single centre experience.

13 patients have been transplanted at Institute of Hematology and Blood Transfusion since 1995 using allogeneic PBPC either alone or with bone marrow as a source of progenitor cells. All donors were G-CSF mobilised HLA identical family members. PBPC harvests were performed on D 4,5, (6) of G-CSF administration. The medium content of TNC, CD34+, CD3+, CD4+and CD8+cells/kg b.w. of the recipients in the grafts were: 13,1x10(8)(TNC), 11,4x10(6)(CD34+), 393x10(6)(CD3+) 243x10(6)(CD4+), 125x10(6)(CD8+) The patients received either BuCy2 or CyTBI preparative regimen and Cyclosporin A + short course of Methotrexate for GVHD prophylaxis. Engraftment of ANC >500 was achieved by D+16 and PLT >20.000 by D+19. Three of ten evaluable patients developed acute and three of nine chronic GVHD. 8 patients survive with the longest follow up 776 days.

Helper T lymphocyte precursor frequency analysis in alloreactivity detection.

The utility of IL-2 secreting helper T lymphocyte precursors (HTLp) frequency testing has been evaluated for detecting alloreactivity. The frequency of HTLp was approached by limiting dilution assay. High HTLp frequency was detected in 20 out of 30 HLA matched unrelated pairs (67%). The comparison of HTLp and CTLp (cytotoxic T lymphocyte precursors) frequencies in HLA matched unrelated pairs showed that the two examinations are not fully alternative in detecting alloreactivity. This could suggest the utility of combined testing of both HTLp and CTLp frequencies for alloreactivity assessment. In contrast, five positive HTLp values were only found among 28 HLA genotypic identical siblings (18%). Previous CTLp limiting dilution studies showed very low or undetectable CTLp frequency results in that group. For that, HTLp assay remains to be the only cellular in vitro technique detecting alloreactivity in these combinations.

Frequency analysis of cytotoxic T lymphocyte precursors in search for donors in bone marrow transplantation.

The usefulness of cytotoxic T lymphocytes precursors (CTLp) frequency analysis in the search for donors in bone marrow transplantation was studied. The frequency of anti-recipient CTLp was approached by limiting dilution assay in HLA matched unrelated, HLA partially matched related and HLA genotypically identical donors. The majority of patients examined were affected with different hematological malignancies. Alloreactive CTLp recognizing non-HLA gene products were not detected in pretransplant examination of two pairs of HLA identical siblings. However, an increased incidence of allospecific CTLp was identified in HLA matched MLC negative unrelated pairs. Thus, CTLp assay allowed to uncover the residual Class I incompatibilities that remained hidden in standard serotyping. In two matched unrelated pairs with high pretransplant CTLp frequency the severe acute graft-versus-host disease (GVHD) developed after bone marrow transplantation. Examination of other relatives in patients lacking an HLA identical sibling showed the importance of Class I incompatibility for CTLp generation as well. The lack of correlation between CTLp frequency and HLA-D disparity could suggest that Class II antigens do not participate in CTLp induction. With one exception we had good correlation between MLC and DNA analysis of Class II antigens demonstrating that MLC gives interpretable results even in unrelated pairs. Our results demonstrate the significance of CTLp frequency assay in detection of residual Class I incompatibilities in matched unrelated pairs and in assessment of Class I compatibility in related pairs. For that it should be used in the final selection of BMT donors.

Cytotoxic T lymphocyte precursor frequency analysis in the selection of HLA matched unrelated donors for hematopoietic stem cell transplantation: the correlation of CTLp frequency with HLA class I genotyping and aGVHD development.

The selection of human leukocyte antigen (HLA) compatible unrelated donors for hematopoietic stem cell transplantation (HSCT) is based on the direct genotyping of HLA class I and class II alleles (HLA-A, -B, -C, -DRB1, -DQB1 loci). The cellular test estimating the frequency of cytotoxic T lymphocyte precursors (CTLp) has been included into the selection procedure of unrelated donors to detect the class I alloreactivity and to predict acute graft versus host disease (aGVHD) occurrence and severity. The relationship between HLA-A, -B, -C high/medium resolution genotyping and CTLp activation was analysed in the cohort of 78 unrelated donor/patient pairs indicated for HSCT. The high frequency of CTLp (> 1:100,000) correlated significantly (p < or = 0.0002) with the incompatibilities in alleles of HLA-A, -B, -C loci. Nevertheless, the results of HLA-A, -B, -C genotyping and CTLp assay are not fully alternative, suggesting that the CTLp test gives its specific information. The high CTLp frequency (CTLpf) in 14/35 pairs fully matched by HLA class-I alleles genotyping could reflect the influence of another factors upon the CTLp activation. On the contrary, the low CTLp frequency values (< or = 1:100,000) found in 8/43 pairs with existing HLA class-I alleles incompatibilities could indicate the immunological permissivity of these particular mismatches. The clinical relevance of the CTLp test for aGVHD prediction has been also analysed. The relationship between CTLp activation in vitro and the incidence and severity of aGVHD was evaluated in 37 patients who underwent allogeneic HSCT. The severe form of aGVHD (grade III-IV) developed in 9 of 18 cases (50%) with the high pretransplant CTLpf value. The patients with the low CTLpf (n = 19) suffered from the severe form of aGVHD in 2 cases (10%) only, the remaining 17 patients from this group were without aGVHD symptoms or developed only the mild form of aGVHD (I-II). The relationship between CTLp results and the incidence and severity of aGVHD was found statistically significant (p < or = 0.01).

Predictive value of HLA class II PCR typing for the outcome of mixed lymphocyte reaction.

The use of HLA-DRB1 and -DQB1 polymerase chain reaction-sequence-specific primer (PCR-SSP) typing at different levels of resolution for MLR prediction was assessed in 54 HLA-A and -B matched donor/recipient unrelated pairs and 89 HLA-A and -B identical siblings. Graft-versus-host (GvH) direction one-way MLR was evaluated unless stated otherwise. The typing of DRB1 alleles satisfactory for MLR prediction in HLA identical siblings (P = 0.0015) does not appear to be sufficient in matched unrelated pairs (P = 0.2407). Using more discriminatory PCR-SSP typing, the disparity in DRB1 allelic subtypes was predominantly found in the category of DRB1 allele compatible, MLR positive unrelated pairs. Besides, DRB1 allelic subtype mismatches were revealed in five of the forty-one DRB1 allele compatible, MLR negative unrelated pairs. More discriminatory typing made the correlation between DRB1 compatibility and MLR negativity extremely significant (P = 0.0001). As for these five exceptional cases, the reciprocal host-versus-graft (HvG) direction MLR was considered, too. This allowed HLA-D disparity to be disclosed in two of them. An uninterpretable result reflecting defective MLR reactivity occurred in one case. Negative reciprocal MLR in the last two DRB1 allelic subtype incompatible pairs is hardly to explain without postulation of MLR silent DRB1 allelic subtype mismatches. An analysis in unrelated pairs showed a role of some DQB1 gene products in the proliferative response too. GvH direction positive MLR was found in two HLA identical siblings among the 89 tested. The DPB1 incompatibility detected in one of them could be a potential cause of proliferative response but MLR reactivity in the other, DPB1 identical, pair cannot be interpreted easily.

The search for HLA-compatible bone marrow donors. Procedure currently used in the Institute of Hematology and Blood Transfusion, Prague.

The search for compatible donors is based on the HLA types of donors and recipients. HLA-A, -B and -DRB1 antigens or alleles must be unequivocally typed in donors and recipients. The typing of HLA-DQB, -DPB and -C gene products is also useful to characterize the HLA phenotype, but it is not absolutely necessary in the donor selection. The standard serological methods using alloantisera and one-dimensional isoelectric focusing are used for the typing of HLA class I antigens. Recently, DNA analysis of class I alleles has been introduced. In HLA class II typing the serological analysis was generally replaced by DNA analysis. In addition to typing techniques that determine the individual HLA alleles or HLA gene products, the cellular matching techniques are used in the selection procedure (mixed lymphocyte culture, cytotoxic T lymphocyte precursor frequency assay, and IL-2-producing helper T lymphocyte precursor frequency assay). The cellular matching techniques determine the compatibility in the regions of HLA comprising several HLA loci; some of them may detect minor histocompatibility (non-HLA) gene disparities as well.

Dimorphic growth and lipase production in lipolytic yeasts.

N-Acetylglucosamine and citrate promoted intensive dimorphic growth and significant lipase synthesis in Yarrowia lipolytica. In contrast, use of a phosphate buffer instead of citrate for buffering the medium stimulated only dimorphic growth. No correlation between dimorphic growth and a high lipase synthesis studied in five different species of lipolytic yeasts was demonstrated. The data provide evidence against the inevitable linkage between the capability (and/or intensity) of mycelium formation and a high level of extracellular lipase in lipolytic yeasts.

Kinetics of delta 5,7-sterol accumulation during growth of Saccharomyces cerevisiae.

Saccharomyces cerevisiae accumulates delta 5,7-sterols up to 4 mg per g biomass. The differential rate of sterol synthesis continually increases during growth, its value only being decreased at sterol levels higher than 30 mg per g biomass. The specific rate of sterol synthesis reaches a broad maximum during the growth phase. The gradual sterol accumulation pattern is dominant in cultures growing both on fermentable and nonfermentable carbon sources and is modulated by glucose repression. Limited feeding with sucrose has a significantly greater negative impact on sterol accumulation than feeding with ethanol as a carbon source.

Effect of growth rate on ethanol tolerance of Saccharomyces cerevisiae.

delta 5,7-Sterol-accumulating Saccharomyces cerevisiae cells growing in chemostat at a specific growth rate of 0.075/h exhibited higher ethanol tolerance measured as ethanol-induced death and anaerobic growth inhibition than the cells growing at 0.2/h, the difference being dependent on the carbon-to-nitrogen molar proportion in the medium. The observed difference in sensitivity to ethanol of anaerobic growth between the slowly and rapidly-growing cells was completely reversed as a result of a block in sterol synthesis causing a negligible synthesis of delta 5,7-sterols. Two physiological parameters, budding frequency and membrane composition, evidently affected ethanol tolerance. Differences between the delta 5,7-sterol-synthesizing and deficient strains documented a profound effect of the quality of the sterol present on the physiological state of the cell.

Effect of clomiphene on the content of sterols and fatty acids in Saccharomyces cerevisiae.

During cultivation of Saccharomyces cerevisiae clomiphene regulates both quantitative and qualitative production of sterols and fatty acids as identified by gas chromatography and mass spectrometry. The content of sterols decreases to 75%, the production of fatty acids is comparable with that in the control. The occurrence of sterols increases; sterols with methyl group in position 4, without double bond in position 22 and with double bond in position 24(25) or 24(28) predominate. Among fatty acids shorter saturated and monoene acids are primarily produced, 2-hydroxy acids practically disappeared.

Ethanol-induced death and lipid composition of Saccharomyces cerevisiae: a comparative study of the role of sterols.

Ethanol tolerance of four Saccharomyces cerevisiae strains characterized by different amounts of delta 5,7-sterols was tested. The individual tolerances did not correlate with the strains sterol levels. The highly and medium-accumulating strains exhibited the highest and lowest ethanol tolerances, respectively.

Effect of ammonium ions on delta 5,7-sterol synthesis in Saccharomyces cerevisiae.

The effect of ammonium concentration in the medium on delta 5,7-sterol synthesis was examined. Higher concentrations of this nitrogen source in the medium decreased sterol synthesis and accumulation during growth. An intermittent supply with ammonium resulted in a proportional synthesis of delta 5,7-sterols and biomass. The carbon to nitrogen molar ratio of greater than or equal to 40 allowed the maximum accumulation of delta 5,7-sterols with our strain of baker's yeast.

[Frequency of cytotoxic and helper T-lymphocyte precursors in donors with alloreactivity to recipient histocompatibility antigens. Possible use in predicting acute graft vs host disease in bone marrow transplantation]. / Frekvence prekurzoru cytotoxických a pomocných T lymfocytu dárce aloreaktivních vuci histokompatibilitním antigenum príjemce. Moznosti vyuzití pro predpoved aGVHD pri transplantaci kostní drene.

BACKGROUND: The utility of cytotoxic T lymphocyte precursor (CTLp) and helper T lymphocyte precursor (HTLp) frequencies estimation for detecting alloreactivity and for the prediction of acute graft versus host disease (aGVHD) has been evaluated. METHODS AND RESULTS: The limiting dilution assay and a maximum likelihood statistical programme were used for CTLp and HTLp frequency estimation. A high CTLp frequency suggesting the presence of hidden class I mismatches was detected in 41.2% of unrelated pairs. HLA-A and -B matched by serological typing and DRB1 and DQB1 matched by DNA analysis. Severe aGVHD (grade III-IV) occurred in all patients of this group who underwent bone marrow transplantation (BMT). In two patients of the three evaluated with low pretransplant CTLp frequency a mild form (grade I) or no aGVHD developed after unrelated BMT. Positive frequency of alloreactive HTLp was found in 50% of HLA matched unrelated pairs. The comparison of CTLp and HTLp values in the same individuals showed that these two methods are not fully alternative in detecting alloreactivity. In the group of HLA identical siblings, 18.7% of positive HTLp results were only found. Besides HLA-DP incompatibilities, the differences in non-HLA genes could cause this alloreactivity. CONCLUSIONS: CTLp assay has a potential for the prediction for aGVHD development following BMT from HLA matched unrelated donors. CTLp results suggest the necessity of more accurate class I typing in these cases. The comparison of CTLp and HTLp frequencies showed that the results can differ in some unrelated donor-recipient BMT pairs suggesting the convenience of simultaneous performing of both assays for the alloreactivity assessment. More cases have to be considered to determine the relationship between pretransplant HTLp frequency and posttransplant aGVHD development in HLA identical siblings.

[Allogenic transplantation of hematopoietic stem cells in the treatment of children with high-risk acute leukemia]. / Alogenní transplantace kmenových bunek krvetvorby v lécbe detí s vysoce rizikovou akutní leukémií.

BACKGROUND: Most children with acute lymphoblastic leukemia (ALL) and increasing number of children with acute myelogenous leukemia (AML) are currently cured with conventional chemotherapy. Despite of this success there is a subset of patients with high-risk features at diagnosis who are predisposed to a very high risk of relapse. Relapse of AML and early bone marrow relapse of ALL can not be cured by conventional chemotherapy. Allogeneic hematopoietic stem cell transplantation (HSCT) is therapeutic option in these children with very high-risk acute leukemia. METHODS AND RESULTS: Between XI/1989-XII/1996 33 children with acute leukemia (ALL: 22, AML: 11) underwent an allogeneic HSCT from HLA identical related donors (HLA-identical sibling: 30, twin: 1, other HLA-identical relative: 2) at the 2nd Dept. of Pediatrics, University Hospital Motol. Median age of our group was 9 years (1.5-19 y.), boys (n = 23) clearly dominated over the girls (n = 10). The resource of stem cells was bone marrow in 31 children, bone marrow and peripheral blood progenitor cells (PBPC) and PBPC in one child respectively. Myeloablative conditioning regimen varied, consisting of total body irradiation and chemotherapy in 21 children and chemotherapy in 12 children. HSCT was performed in first complete remission of acute leukemia in 9 children (AML: 7, ALL: 2), in second remission in 14 children (AML: 2, ALL: 12), in third remission in 4 children (ALL: 4). Six children underwent HSCT in first partial remission (n = 1) and in second (n = 4) or third (n = 1) chemoresistant relapse. Seven (21%) children died due to post-transplant complications. Nine (28%) children suffered from clinically significant acute graft-versus-host reaction (GVH) and 15% (4/27) children who survived 100 days post-transplant suffered from chronic GVH disease. Relapse of leukemia was diagnosed in 39% (12/31) children. Fourteen (42%) children are alive and well in continuous remission with median follow-up 42 months. CONCLUSIONS: Allogeneic HSCT can cure children with very high-risk acute leukemia in the situations where conventional chemotherapy fails. Relapse of leukemia and GVH reaction are most important causes of post-transplant morbidity and mortality.

[Does insufflation of the abdomen in laparoscopic surgery affect acid-base and ventilatory parameters?]. / Ovlivnuje insuflace bricha pri laparoskopické operaci acidobazické a ventilacní parametry?

The cardiopulmonary and metabolic changes experienced by patients undergoing laparoscopic cholecystectomy with CO2 pneumoperitoneum are not well understood. The purpose of this study is to determine changes of basal parameters during laparoscopy and evaluate their prognostic value. One hundred patients (26 obese, 39 older than 60 years, 7 obese and older than 60) undergoing laparoscopic cholecystectomy for uncomplicated cholecystolithiasis were included in the study. Arterial blood gases, respiratory and ventilatory parameters, heart rate, blood pressure were determined before the induction of pneumoperitoneum, at the peak of operation and after exsufflation. The obtained variables were statistically evaluated. Pneumoperitoneum caused significant hypercapnia and a decrease of pH accompanied with increase of expiratory CO2 concentration, which continued after exsufflation (p < 0.001). The changes were more expressed in older and obese patients and were solely of a respiratory type. No significant changes were observed in the heart rate, blood pressure, minute ventilation, PaO2, SaO2, base excess. Although changes were highly significant, there was no impact on clinical status--all patients survived without problems. The authors conclude that observed increase of carbon dioxide levels and decrease of pH had no impact on survival of patients. Changes were caused mostly by CO2 absorption from the abdominal cavity. Laparoscopic cholecystectomy is a safe and effective procedure even in older and obese patients, especially when insufflation is as low as possible.