Dynamics of specific ammonia-oxidizing bacterial populations and nitrification in response to controlled shifts of ammonium concentrations in wastewater.

Ammonia-oxidizing bacteria (AOB) are essential for the nitrification process in wastewater treatment. To retain these slow-growing bacteria in wastewater treatment plants (WWTPs), they are often grown as biofilms, e.g., on nitrifying trickling filters (NTFs) or on carriers in moving bed biofilm reactors (MBBRs). On NTFs, a decreasing ammonium gradient is formed because of the AOB activity, resulting in low ammonium concentrations at the bottom and reduced biomass with depth. To optimize the NTF process, different ammonium feed strategies may be designed. This, however, requires knowledge about AOB population dynamics. Using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy, we followed biomass changes during 6 months, of three AOB populations on biofilm carriers. These were immersed in aerated MBBR tanks in a pilot plant receiving full-scale wastewater. Tanks were arranged in series, forming a wastewater ammonium gradient mimicking an NTF ammonium gradient. The biomass of one of the dominating Nitrosomonas oligotropha-like populations increased after an ammonium upshift, reaching levels comparable to the high ammonium control in 28 days, whereas a Nitrosomonas europaea-like population increased relatively slowly. The MBBR results, together with competition studies in NTF systems fed with wastewater under controlled ammonium regimes, suggest a differentiation between the two N. oligotropha populations, which may be important for WWTP nitrification.

Galactolipid deficiency in the early pathogenesis of neuronal ceroid lipofuscinosis model Cln8mnd : implications to delayed myelination and oligodendrocyte maturation.

AIMS: CLN8 deficiency underlies one of a group of devastating childhood neurodegenerative disorders, the neuronal ceroid lipofuscinoses. The function of the CLN8 protein is currently unknown, but a role in lipid metabolism has been proposed. In human CLN8 diseased brains, alterations in lipid composition have been detected. To further investigate the connection of CLN8 to lipid metabolism, we characterized the lipid composition of early symptomatic Cln8-deficient mouse (Cln8(mnd)) brains. METHODS: For lipid profiling, Cln8(mnd) cerebral cortical tissue was analysed by liquid chromatography/mass spectrometry. Galactolipid synthesis was measured through enzyme activity and real-time mRNA expression analyses. Based on the findings, myelination and white matter integrity were studied by immunohistochemistry, stereological methods, electron microscopy and magnetic resonance imaging. The development of myelin-forming oligodendrocytes was also studied in vitro. RESULTS: Sphingolipid profiling showed a selective reduction in myelin-enriched galactolipids. The mRNA expression and activity of UDP-galactose:ceramide galactosyltransferase (CGT), the key enzyme in the galactolipid synthesis, was reduced in the Cln8(mnd) brain. Expression of oligodendrocyte markers suggests a maturation defect. The amount of myelin was reduced in 1-month-old Cln8(mnd) mice, but reached normal levels by 5 months of age. The level of Cln8 gene expression followed the developmental pattern of myelin formation and was high in primary oligodendrocytes. CONCLUSIONS: Taken together, these observations suggest that galactolipid deficiency and delayed myelin maturation characterize the early CLN8 disease pathogenesis through a maturation defect of oligodendrocytes.

Association of ABCB1 polymorphisms with survival and in vitro cytotoxicty in de novo acute myeloid leukemia with normal karyotype.

Overexpression of the multi-drug transporter P-glycoprotein, encoded by the ABCB1 gene, is a clinically relevant problem in acute myeloid leukemia (AML). Polymorphisms in ABCB1 might contribute to cancer risk and therapeutic response. We therefore investigated the influence of polymorphisms G1199A, C1236T, G2677T/A and C3435T on cancer susceptibility, in vitro cytotoxicity and overall survival in 100 de novo AML patients with normal karyotype. Patients with 1236C/C or 2677G/G genotypes showed poorer survival than patients with other genotypes (P=0.03 and P=0.02, respectively). Both these genotypes were significant factors for survival in multivariate analysis, along with age, NPM1 and FLT3 mutation status. In vitro cytotoxicity studies demonstrated that leukemic cells from 1236T/T and 2677T/T patients were significantly more susceptible to mitoxantrone (P=0.02), and tended to be more susceptible to etoposide and daunorubicin (P=0.07-0.09), but not to cytarabine. No significant difference in allele frequencies was found between patients and healthy volunteers (n=400).

Nitrification potential and population dynamics of nitrifying bacterial biofilms in response to controlled shifts of ammonium concentrations in wastewater trickling filters.

Nitrogen removal in wastewater treatment is energy consuming and often carried out in biofilm nitrifying trickling filters (NTFs). We investigated nitrification potential and population dynamics of nitrifying bacteria in pilot-plant NTFs fed with full-scale plant wastewater with high (8-9 mg NH(4)(+)l(-1)) or low (<0.5mg NH(4)(+)l(-1)) ammonium concentrations. After ammonium shifts, nitrification potentials stabilized after 10-43 days depending on feed regime. An NTF fed with 3 days of high, and 4 days of low load per week reached a high nitrification potential, whereas a high load for 1 day a week gave a low potential. Nitrosomonas oligotropha dominated the AOB and changes in nitrification potentials were not explained by large population shifts to other AOBs. Although nitrification potentials were generally correlated with the relative amounts of AOB and NOB, this was not always the case. Ammonium feed strategies can be used to optimize wastewater treatment performance.

Smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in BAL cells.

OBJECTIVES: A gene-environment interaction between HLA-DR shared epitope genes and smoking in anti-cyclic citrullinated peptide antibody-positive rheumatoid arthritis (RA) has been reported. Identification of citrullinated proteins in bronchoalveolar lavage (BAL) cells from smokers has led to the suggestion that citrullination induced by smoking might be the first step in the pathogenic chain of RA. OBJECTIVE: To confirm and extend these findings. METHODS: Immunohistochemistry was performed on BAL cells and bronchial mucosal biopsy sections obtained through bronchoscopy from 14 healthy smokers and 16 healthy non-smokers. Two antibodies recognising citrullinated proteins, two antibodies recognising peptidylarginine deiminase (PAD)2 enzyme and one recognising PAD4 enzyme were used. RESULTS: Citrullinated proteins are upregulated in BAL cells of healthy smokers compared with healthy non-smokers. This was associated with higher expression of the PAD2 enzyme. The same level of citrullinated proteins was present in bronchial mucosal biopsy specimens of healthy smokers and non-smokers, despite higher expression of PAD2 in smokers. CONCLUSION: This study provides evidence that smoking enhances PAD2 expression in the bronchial mucosal and alveolar compartment, with consequent generation of citrullinated proteins in the latter. Smoking is an environmental factor that may lead to citrulline autoimmunity in genetically susceptible subjects.

Acyl chain-dependent effect of lysophosphatidylcholine on human neutrophils.

Lysophosphatidylcholine (LPC) is the most abundant lysophospholipid in plasma and tissues, and its level increases in ischemia and inflammation. LPC induces various proinflammatory actions in leukocytes, endothelial cells, and smooth muscle cells, but its effects may vary, depending on the acyl chain. In the present study, we identified the molecular species of LPC in human plasma and studied their effects on human neutrophils. Unsaturated LPC species over a wide concentration range (5-200 microM) induced long-lasting superoxide production in neutrophils. The response was preceded by a >10-min lag time and lasted for 60-90 min. Superoxide production was prevented when albumin was added together with LPC at a molar ratio of 1:2 or higher, and significant inhibition was observed even when albumin was added 4-8 min after LPC. Saturation of albumin by fivefold molar excess of stearic acid reduced the inhibitory effect significantly. Saturated LPCs, particularly the most abundant 16:0 species, induced significantly less superoxide production than the unsaturated species and only at 5-10 microM concentrations. Saturated LPC species elicited a several-fold higher increase in cytoplasmic calcium and at >20 microM, increased plasma membrane permeability. A mixture of LPCs mimicking the plasma LPC composition induced nearly similar superoxide production as the most active LPC18:1 alone. These results indicate remarkable acyl chain-dependent differences in the cellular effects of LPC. Elevation of LPC level may increase inflammation through activation of neutrophil NADPH oxidase, particularly when the simultaneous increase of free fatty acids diminishes the ability of albumin to scavenge LPCs.

Removal of geosmin and MIB by biofiltration--an investigation discriminating between adsorption and biodegradation.

Geosmin and 2-methylisoborneol (MIB) are two substances causing earthy/musty odours that are difficult to remove by conventional chemical drinking water treatment. In this study removal of geosmin and MIB by biofiltration of untreated surface water was investigated using granular activated carbon (GAC) and crushed expanded clay (EC) as filter media. Biofiltration through both GAC and EC removed geosmin and MIB present at low (20 ng l(-1)) concentrations by at least 97% at an empty bed contact time of 30 minutes and a temperature of 15 degrees C. At lower temperature (6-12 degrees C) and simultaneously lower biomass concentrations, removal efficiency was similar in the GAC but considerably lower in the EC biofilter, pointing to a second mechanism different from biodegradation. Consequently, microbial activity was suppressed with azide to enable discrimination between biodegradation and adsorption. During azide dosage, the GAC biofilters still removed geosmin and MIB nearly unaffectedly. In the EC biofilter, however, removal of both odorants ceased completely. Methylene blue adsorption confirmed that the GAC, even after almost four years of operation receiving surface water, had capacity to remove geosmin and MIB by adsorption. Since odour episodes commonly occur during the warm season when microbiological activity is high, EC constitutes a viable option as carrier medium for direct biological filtration of surface water. The additional GAC adsorption capacity however adds robustness to the removal process.

Performance of direct biofiltration of surface water for reduction of biodegradable organic matter and biofilm formation potential.

Direct biofiltration of surface water may be considered as pre-treatment for membrane filtration, in order to reduce fouling. The biofiltration process was investigated with regard to biodegradable organic matter and biofilm formation, covering the annual variations under moderately cold climate conditions. Granular activated carbon (GAC) and two types of crushed expanded clay (EC) were compared as filter media. To assess the biological properties of the biofilters, viable biomass and respiratory activity was examined. Biofiltration removed assimilable organic carbon (AOC) and biodegradable dissolved organic carbon (BDOC) by about 30% and also reduced the bacterial concentration in the water phase. Also, biofilm formation in the treated water was reduced by 80-90% during summer and winter conditions. The reductions in the investigated parameters were similar in the biofilters with GAC and EC. Likewise, a similar amount and development of biomass was found in the GAC and EC biofilters of comparable grain size with a pronounced stratification from top to bottom of the filter bed. The specific respiratory activity of the biofilter biomass was dependent on raw water temperature. Even though slight correlations between BDOC removal and temperature or respiratory activity were observed, AOC and BDOC removals were mainly dependent on the feed water concentrations of these compounds. The results indicate that direct biofiltration of surface water, by reducing AOC, BDOC and biofilm formation in the water, may be an advantageous pre-treatment for membrane filtration processes.

Influence of flocculation and settling properties of activated sludge in relation to secondary settler performance.

Floc characteristics were studied at a full scale activated sludge treatment plant with a unique process solution incorporating pre-denitrification with post-nitrification in nitrifying trickling filters. Since greater nitrogen removal is achieved when more secondary settled wastewater is recirculated to the trickling filters, the secondary settlers are always operated close to their maximal capacity. The flocculation and settling properties are therefore crucial and have an effect on the overall plant performance. Since the plant is operated at a short sludge age, these properties change quickly, resulting in variable maximal secondary settler capacity. The dynamics in floc structure and microbial community composition were studied and correlated to the secondary settler performance. Fluorescence in situ hybridisation was used to investigate the microbial community structure and their spatial distribution. The floc structure could to some extent be related to the flocculation and settling properties of the sludge. Even small differences had an influence suggesting that colloidal properties also play a significant role in determining the floc properties. No correlation between microbial community composition and settling properties could be established with the group-specific probes investigated.

Community survey of ammonia-oxidizing bacteria in full-scale activated sludge processes with different solids retention time.

AIMS: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. METHODS AND RESULTS: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. CONCLUSIONS: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all beta-proteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used.

Formation of helical hairpins during membrane protein integration into the endoplasmic reticulum membrane. Role of the N and C-terminal flanking regions.

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a common structural element in integral membrane proteins. Previous studies on the sequence determinants of helical hairpin formation have focussed on the role of polar and charged residues placed centrally in a long stretch of hydrophobic residues, and have yielded a "propensity scale" for the relative efficiency with which different residues promote the formation of helical hairpins. In this study, we shift our attention to the role of charged residues flanking the hydrophobic stretch. Clusters of charged residues are known to hinder membrane translocation, and thus flanking charged residues may conceivably force a long hydrophobic segment to form a helical hairpin even if there are no or only weakly turn-promoting residues in the hydrophobic stretch. We indeed find that Lys and, more surprisingly, Asp residues strongly affect helical hairpin formation when placed next to a poly-Leu-based transmembrane segment. We also find that a cluster of four consecutive Lys residues can affect the efficiency of helical hairpin formation even when placed approximately 30 residues downstream of the hydrophobic stretch. These observations have interesting implications for the way we picture membrane protein topogenesis within the context of the endoplasmic reticulum (ER) translocon.

Adhesion of type 1-fimbriated Escherichia coli to abiotic surfaces leads to altered composition of outer membrane proteins.

Phenotypic differences between planktonic bacteria and those attached to abiotic surfaces exist, but the mechanisms involved in the adhesion response of bacteria are not well understood. By the use of two-dimensional (2D) polyacrylamide gel electrophoresis, we have demonstrated that attachment of Escherichia coli to abiotic surfaces leads to alteration in the composition of outer membrane proteins. A major decrease in the abundance of resolved proteins was observed during adhesion of type 1-fimbriated E. coli strains, which was at least partly caused by proteolysis. Moreover, a study of fimbriated and nonfimbriated mutants revealed that these changes were due mainly to type 1 fimbria-mediated surface contact and that only a few changes occurred in the outer membranes of nonfimbriated mutant strains. Protein synthesis and proteolytic degradation were involved to different extents in adhesion of fimbriated and nonfimbriated cells. While protein synthesis appeared to affect adhesion of only the nonfimbriated strain, proteolytic activity mostly seemed to contribute to adhesion of the fimbriated strain. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, six of the proteins resolved by 2D analysis were identified as BtuB, EF-Tu, OmpA, OmpX, Slp, and TolC. While the first two proteins were unaffected by adhesion, the levels of the last four were moderately to strongly reduced. Based on the present results, it may be suggested that physical interactions between type 1 fimbriae and the surface are part of a surface-sensing mechanism in which protein turnover may contribute to the observed change in composition of outer membrane proteins. This change alters the surface characteristics of the cell envelope and may thus influence adhesion.

Formation of cytoplasmic turns between two closely spaced transmembrane helices during membrane protein integration into the ER membrane.

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a recurring structural element in integral membrane proteins, and may serve as a compact unit that inserts into the membrane en bloc. Previously, we have determined the propensities of the 20 natural amino acids, when present in the middle of a long hydrophobic stretch, to induce the formation of a helical hairpin with a lumenally exposed turn during membrane protein assembly into the endoplasmic reticulum membrane. Here, we present results from a similar set of measurements, but with the turn placed on the cytoplasmic side of the membrane. We find that a significantly higher number of turn-promoting residues need to be present to induce a cytoplasmic turn compared to a lumenal turn, and that, in contrast to the lumenal turn, the positively charged residues Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns.

Surgical approach and prognostic factors after peptic ulcer perforation.

OBJECTIVE: To find out which prognostic factors were important in predicting postoperative mortality and length of hospital stay in patients with perforated peptic ulcers. DESIGN: Retrospective study. SETTING: Teaching hospital, Lund, Sweden. SUBJECTS: 246 patients with perforated peptic ulcer who presented between January 1974 and December 1992. INTERVENTION: Cox proportional hazards analysis. MAIN OUTCOME MEASURES: Influence of age, sex, coexisting disease, duration of symptoms, site of perforation and operative technique on mortality and length of hospital stay. RESULTS: Age over 75 years (p = 0.002), coexisting cardiac or pulmonary disease (p = 0.02), perforation of the cardia or body of the stomach (p = 0.02), lapse of more than 12 hours between start of symptoms and operation (p = 0.006) and type of operation (p < 0.0001) had a significant influence on hospital mortality. Age over 75 years (p < 0.0001) and lapse of more than 12 hours between start of symptoms and operation (p = 0.03) significant influenced the likelihood of a prolonged stay in hospital. CONCLUSION: Patients with perforated peptic ulcers should be operated on as soon as possible. Simple closure is simple and safe with relatively low mortality and short stay in hospital.

Effect of ionic strength on initial interactions of Escherichia coli with surfaces, studied on-line by a novel quartz crystal microbalance technique.

A novel quartz crystal microbalance (QCM) technique was used to study the adhesion of nonfimbriated and fimbriated Escherichia coli mutant strains to hydrophilic and hydrophobic surfaces at different ionic strengths. This technique enabled us to measure both frequency shifts (Deltaf), i.e., the increase in mass on the surface, and dissipation shifts (DeltaD), i.e., the viscoelastic energy losses on the surface. Changes in the parameters measured by the extended QCM technique reflect the dynamic character of the adhesion process. We were able to show clear differences in the viscoelastic behavior of fimbriated and nonfimbriated cells attached to surfaces. The interactions between bacterial cells and quartz crystal surfaces at various ionic strengths followed different trends, depending on the cell surface structures in direct contact with the surface. While Deltaf and DeltaD per attached cell increased for nonfimbriated cells with increasing ionic strengths (particularly on hydrophobic surfaces), the adhesion of the fimbriated strain caused only low-level frequency and dissipation shifts on both kinds of surfaces at all ionic strengths tested. We propose that nonfimbriated cells may get better contact with increasing ionic strengths due to an increased area of contact between the cell and the surface, whereas fimbriated cells seem to have a flexible contact with the surface at all ionic strengths tested. The area of contact between fimbriated cells and the surface does not increase with increasing ionic strengths, but on hydrophobic surfaces each contact point seems to contribute relatively more to the total energy loss. Independent of ionic strength, attached cells undergo time-dependent interactions with the surface leading to increased contact area and viscoelastic losses per cell, which may be due to the establishment of a more intimate contact between the cell and the surface. Hence, the extended QCM technique provides new qualitative information about the direct contact of bacterial cells to surfaces and the adhesion mechanisms involved.

A turn propensity scale for transmembrane helices.

Using a model protein with a 40 residue hydrophobic transmembrane segment, we have measured the ability of all the 20 naturally occurring amino acids to form a tight turn when placed in the middle of the hydrophobic segment. Turn propensities in a transmembrane helix are found to be markedly different from those of globular proteins, and in most cases correlate closely with the hydrophobicity of the residue. The turn propensity scale may be used to improve current methods for membrane protein topology prediction.

Does desmopressin reduce bleeding in liver surgery? An experimental study on pigs.

The main problem with liver surgery is to control intraoperative bleeding. Bleeding together with postoperative infection and liver failure are the most common causes of postoperative morbidity and mortality in liver surgery. Hospitals with experience of liver resection, report postoperative mortality rates of 3.2%-14.3%. Improved results in liver surgery have been made by refinement of surgical methods and modern techniques. Desmopressin acetate (Octostim) reduces bleeding time by activating platelets and increasing the level of coagulation factor VIII. The purpose with this study was to see if desmopressin could reduce bleeding in a standardized experimental liver resection. Sixteen Swedish domestic pigs were randomized to receive desmopressin 0.3 ug/kg i.v. or placebo, and underwent liver resection with conventional finger fracture technique. The intraoperative blood loss was estimated by several parameters such as pre and postoperative haemoglobin values and by weighing the number of gauze swabs used. One week postoperatively new haemoglobin samples were taken and the pigs were killed. At autopsy the resection area was inspected. All sixteen pigs survived the operation and the postoperative week. There was no significant difference in blood loss between the desmopressin and placebo group. In conclusion, our study showed that desmopressin did not reduce intraoperative bleeding in experimental liver resection.

In Situ Detection of High Levels of Horizontal Plasmid Transfer in Marine Bacterial Communities.

Gene transfer of the conjugative plasmid pBF1 from Pseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C. Dahlberg et al., Mol. Biol. Evol. 15:385-390, 1998). pBF1 was tagged with the gfp gene controlled by a lac promoter which is down regulated in the donor cell by a chromosomal repressor (lacIq). The plasmid donor cells (Pseudomonas putida KT2442) subsequently do not express gfp. Transfer to recipient strains lacking the repressor results in expression of gfp. The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level. By using this method, transfer of pBF1::gfp and expression of the gfp gene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater. Second, we measured transfer of pBF1 from P. putida to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria. Plasmid transfer was detected on surfaces and in bulk seawater. Seawater bacteria with different morphologies were shown to receive the plasmid. Gene transfer frequencies of 2.3 x 10(-6) to 2.2 x 10(-4) transconjugants per recipient were recorded after 3 days of incubation.

Growth hormone treatment prevents the decrease in insulin-like growth factor I gene expression in patients undergoing abdominal surgery.

Acquired GH resistance together with reduced skeletal muscle mass are found in patients with increased protein catabolism due, for example, to sepsis, trauma, or major surgery. Both administration of glutamine-containing parenteral nutrition and GH treatment have been found to diminish this catabolism. The effects of GH are mediated in part by insulin-like growth factor I (IGF-I) that is produced in the liver and locally in GH target tissues. The aim of this study was to investigate the effect of GH treatment on expression of the IGF-I gene and GH receptor (GHR) gene in skeletal muscle after major surgery. A new quantitative RT-PCR-based assay was established to measure IGF-I gene expression. Metabolically healthy patients, without significant preoperative weight loss, who were undergoing elective abdominal surgery were included in the study. Five patients (one woman and four men) were treated with daily injections of GH (0.3 IU/kg.day) in addition to being given total parenteral nutrition including glutamine (0.28 g/kg.day). The control group consisted of eight patients (three women and five men), who were given glutamine-enriched total parenteral nutrition but no GH. A muscle biopsy was taken from the lateral portion of the quadriceps femoris muscle preoperatively (day 0) after induction of anesthesia. A second biopsy was taken under local anesthesia on postoperative day 3. Total ribonucleic acid (RNA) was extracted from the muscle biopsies, and IGF-I messenger RNA (mRNA) and GHR mRNA were measured by competitive quantitative RT-PCR assays. IGF-I mRNA and GHR mRNA levels were related to the expression of a housekeeping gene (cyclophilin). In the control group, IGF-I mRNA levels decreased from 1505 +/- 265 (mean +/- SEM) transcripts/cpm cyclophilin on day 0 to 828 +/- 172 on day 3 (P < 0.05). In contrast, IGF-I mRNA levels did not change in the GH-treated group (1188 +/- 400 transcripts/cpm cyclophilin on day 0 vs. 1089 +/- 342 transcripts/cpm cyclophilin on day 3). No statistically significant changes were seen in GHR expression. We conclude that administration of GH prevents the reduction in IGF-I gene expression in skeletal muscle after abdominal surgery.

A FISH cosmid 'cocktail' for detection of 13q deletions in chronic lymphocytic leukaemia--comparison with cytogenetics and Southern hybridization.

The most frequent structural chromosome abnormality in chronic lymphocytic leukaemia (CLL) is deletion at chromosome 13q14. Studies with Southern blot hybridisation have revealed deletions in the region located telomeric of the retinoblastoma gene in more than 40% of cases. The highest frequency of homozygous deletions has been found at the D13S319 locus and it is likely that a new tumour suppressor gene is located close to this region. We have analysed deletions in the D13S319 region in 20 selected CLL patients using conventional cytogenetic analysis, fluorescence in situ hybridisation (FISH) and Southern blot hybridisation. FISH and Southern hybridisation are equally efficient in detecting deleted clones in our study. However, FISH analysis indicate that subclones with different numbers of alleles in the D13S319 region can exist simultaneously. The cytogenetic analyses confirm that clones with different chromosomal abnormalities can occur in patients with CLL and that 13q14 deletions can be limited to one of these subclones. Furthermore, the FISH analyses show that trisomy 12 and deletion of 13q14 can occur in the same cell clone. Finally, our study confirms that mitogen stimulation of peripheral blood cells from CLL patients before FISH analysis may result in a sharp increase in normal appearing cells, which can hide leukaemic clones with deletions in the D13S319 region.