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1. The use of flowering vegetation has been widely advocated as a strategy for providing parasitoids and predators with nectar and pollen. However, their herbivorous hosts and prey may exploit floral food sources as well. 2. Previous laboratory studies have shown that not all flower species are equally suitable in providing accessible nectar. Relatively little is known about actual nectar exploitation under field conditions. 3. The present study investigates nectar exploitation by the pest, Plutella xylostella, and its parasitoid, Diadegma semiclausum, under field conditions and examines whether floral nectar exploitation in the field can be predicted based on controlled laboratory studies. 4. Insects were collected from fields bordered by flowering margins containing Fagopyrum esculentum, Lobularia maritima, Anethum graveolens, Centaurea jacea or the grass Lolium perenne (control). Whole insect bodies were individually assayed by HPLC to establish their sugar profile as a measure of the level of energy reserves and the degree of food source use. 5. The average overall sugar content of P. xylostella and D. semiclausum collected in fields bordered by flowering margins was significantly higher than those of individuals collected from grass-bordered control plots. To the authors' knowledge, this represents the first demonstration that nectar-providing plants enhance the energetic state of herbivores under field conditions. 6. In contrast to earlier laboratory studies, the present study did not find elevated sugar contents in P. xylostella and D. semiclausum individuals collected from fields bordered by buckwheat (F. esculentum). 7. The present study shows widespread sugar feeding by both the herbivore and its parasitoid. It also shows that laboratory studies establishing nectar exploitation under controlled conditions can not always be extrapolated to actual exploitation under field conditions. This emphasises the importance of studying field-collected insects with regard to food source use and nutritional status.

Extracts of VISCUM ALBUM L. produced by a specific homogenization procedure contain viscotoxins (VT) and liposome-like membrane vesicles, formed from cellular membranes. Interactions between these membrane structures and viscotoxins are characterized in this work. Binding properties of viscotoxins with mistletoe extracts or isolated membrane vesicles were analyzed by gel permeation chromatography (GPC) and centrifugation, followed by HPLC/UV for viscotoxin detection. The experiments show that a part of the viscotoxins is bound to membrane vesicles, and that this binding to the membrane structures is reversible. In the case of the vesicles studied from an extract of 100 mg plant material per mL (0.30 mM phospholipids, 244 μg/mL VT), 64 μg/mL VTs are bound to the membranes. The binding properties of the viscotoxin isoforms are different. VTA3 clearly binds more intensively to membrane structures than VTA2 or VTA1. Possible interaction of viscotoxins with DNA, which is also discussed as a mechanism of viscotoxin action, could be shown to be negligible in the framework of these experiments. CV:coefficient of variation DNA:deoxyribonucleic acid F:fraction GPC:gel permeation chromatography HPLC:high performance liquid chromatography HTH:helix-turn-helix motif PL:phospholipid SPE:solid phase extraction VT:viscotoxin

During the preparation of plant extracts by a press-slit technique, membranes of cell walls and cell organelles of the plant material form vesicles, which are colloidally dispersed. It was assumed that chlorophyll-containing green extracts enclose lipoidic structures. Vesicles in aqueous mistletoe extracts (extracts of Viscum album L.) were analyzed by cryo-transmission electron microscopy (cryo-TEM) without fixation. For the first time, it was possible to analyze unfixed vesicles in the mistletoe extract. Micrographs of cryo-TEM showed predominantly unilamellar vesicles of different sizes. The quantification of vesicles was established through the analysis of phospholipids, which are major components of membranes. The method was validated mainly according to ICH guidelines for the validation of analytical methods (Q2A and Q2B). For further characterization of the vesicle size, a method was developed which is based on the separation of the vesicles from low molecular weight substances by size exclusion chromatography. Fractions were collected and average sizes were determined by multi-angle laser light scattering (MALLS). Furthermore, the UV-vis absorbance and phospholipid concentration were analyzed. Phospholipid quantification was in agreement with photometrical data. Sizes determined by cryo-TEM and by light scattering showed consistent results.

Detection of chimerism by PCR analysis of short tandem repeats (STR) in blood samples of patients who received allogeneic bone marrow transplantation (BMT) has proved to be an important method for early detection of relapse. The prerequisite for this type of analysis is knowledge of donor and recipient pretransplantation genotypes. In some cases, recipient cells from time points prior to BMT are not available and the pretransplant fingerprint cannot be determined. As BM recipients only alter their genotype in blood cells, we attempted to identify patient's pretransplantation genotypes after transplantation in mouthwash samples that contain easily accessible epithelial cells. Of 17 patients who had undergone BMT between one week and 45 months prior to analysis, DNA was isolated from mouthwash cell pellets or from epithelial cells obtained from mouthwashes. PCR analysis of STR loci in the von Willebrand and the tyrosine hydroxylase genes were performed. Even though the mouthwash cell pellets contained about 75% epithelial cells (presumably of recipient origin) and only about 25% leukocytes (presumably of donor origin), three of five patients showed donor genotype and only two patients exhibited chimeric DNA patterns, when cellular DNA was obtained by boiling of mouthwash cell pellets. Following phenol/chloroform extraction, eight of 10 DNA samples exhibited a chimeric pattern, while two of 10 DNAs showed only donor genotype. Of three patients, epithelial cells were attached to magnetic beads prior to DNA isolation. Even this DNA contained donor and recipient material. From our results it appears that blood cells serve as preferential DNA source in mouthwash samples and cannot be removed by epithelial cell separation.

Linear and convergent routes for the large-scale preparation of the hematoregulatory nonapeptide (Glp-Glu-Asp)2-DAS-(Lys)2 (2, SK&F 107647) were investigated. A convergent approach ('3 + 2'-route employing Boc-and benzyl ester protecting groups) was selected for the preparation of multihundred-gram quantities of 2. Key steps were the preparation and the coupling of tripeptide hydrochloride (HCl.H)2-DAS-(Lys(Z)-OBn)2 (6, DAS-2,7-L,L-diaminosuberic acid) and tripeptide Glp-Glu(OBn)-Asp(OBn)-OH (26). Several coupling reagents were investigated in order to reduce the amount of epimerization of this fragment coupling. TDBTU [O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl-1,1,3,3-tetrameth yluronium tetrafluoroborate] was identified as the condensation reagent of choice. Using this synthetic route > 97% pure final product in an overall yield of 35% calculated on di-Boc protected 2,7-L,L-diaminosuberic acid was prepared.

The expression of the natural killer (NK) cell marker CD56 has been reported to occur in NK cell lymphomas/leukemias and a small group of peripheral T-cell lymphomas but has not been studied extensively in primary intestinal non-B-cell lymphomas. Normal human jejunal intraepithelial lymphocytes (IELs) are mainly T-cell receptor (TCR)-αβ + CD3 + CD8 + CD5 low and include an ∼15% fraction of CD56 + cells that could be the cells of origin for CD56 + intestinal T-cell lymphoma (ITL). To test this hypothesis, 70 cases diagnosed as ITL were immunophenotyped, and 15 CD56 + cases (21%) were identified. The majority of the CD56 + lymphomas was of monomorphic small to medium-sized histology, shared the common phenotype βF1 ± CD3e/cyt + CD8 + CD4 − CD5 − CD57 − TIA-1 + and had clonally rearranged TCR γ-chain genes. In contrast, the CD56 − lymphomas were mainly composed of pleomorphic medium and large cells or had a morphology most consistent with anaplastic large-cell lymphoma and were mostly CD8 − . These findings suggest that the majority of CD56 + intestinal lymphomas are morphologically and phenotypically distinct T-cell lymphomas most likely derived from activated cytotoxic CD56 + CD8 + IELs. Some overlapping histological and clinical features between CD56 + and CD56 − ITLs indicate that the former belong to the clinicopathological entity of ITL. The consistent expression of cytotoxic-granule-associated proteins introduces ITL (both CD56 + and CD56 − ) into the growing family of usually aggressive extranodal lymphomas of cytotoxic T-cell and NK-cell derivation. In contrast to putative NK-cell lymphoma of the sinonasal region, intestinal NK-cell lymphoma seems to be very rare.

The yield of protected 2,7- diaminosuberic acid (DAS) prepared by Kolbe reaction of N-protected α-glutamic acid esters is dependent on solvent, temperature, concentration of carboxylate anion, and protecting groups. The highest yield of protected L,L-2,7-diaminosuberic acid is obtained with Boc-Glu-OMe as starting material using MeOH/pyridine/NaOMe as electrolyte.

We describe a multiplex polymerase chain reaction (PCR) method suitable for the detection of all T-cell receptor (TCR) gamma-chain gene rearrangements in patients with lymphoproliferative diseases. 40 patients with various lymphoproliferative disorders and 40 healthy individuals were tested. Clonal TCR gamma rearrangements were identified in all patients with malignant disorders, and in one of 10 cases with established reactive lymphocytosis but not in normal controls. In all individuals testing positive, the patient's specific V and J segment involved in the rearrangement could be determined by simply splitting the multiplex primer mix. Our data show that the multiplex PCR technique enables rapid, simple and sensitive screening for clonal TCR gamma chain gene rearrangements.

A signaling or S-helix has been identified as a conserved, up to 50-residue-long segment in diverse sensory proteins (1). It is present in all major bacterial lineages and in euryarchea and eukaryotes (1). A bioinformatic analysis shows that it connects upstream receiver and downstream output domains, e.g. in histidine kinases and bacterial adenylyl cyclases. The S-helix is modeled as a two-helical parallel coiled coil. It is predicted to prevent constitutive activation of the downstream signaling domains in the absence of ligand-binding (1). We identified an S-helix of about 25 residues in the adenylyl cyclase CyaG from Arthrospira maxima. Deletion of the 25 residue segment connecting the HAMP and catalytic domains in a chimera with the Escherichia coli Tsr receptor changed the response to serine from inhibition to stimulation. Further examination showed that a deletion of one to three heptads plus a presumed stutter, i.e. 1, 2, or 3 × 7 + 4 amino acids, is required and sufficient for signal reversion. It was not necessary that the deletions be continuous, as removal of separated heptads and presumed stutters also resulted in signal reversion. Furthermore, insertion of the above segments between the HAMP and cyclase catalytic domains similarly resulted in signal reversion. This indicates that the S-helix is an independent, segmented module capable to reverse the receptor signal. Because the S-helix is present in all kingdoms of life, e.g. in human retinal guanylyl cyclase, our findings may be significant for many sensory systems.

Although mast cells are hematopoietic cells, little is known about the origin of their precursors in vivo. In this study, the origin (donor v recipient genotype) of human mast cells (MCs) was analyzed in a patient who underwent allogeneic bone marrow transplantation (BMT). The patient presented with secondary acute myeloid leukemia (French-American- British classification, M2) arising from refractory anemia with excess of blast cells and bone marrow (BM) mastocytosis. Transplantation was performed in chemotherapy-induced complete remission. On days 88, 126, 198, and 494 after BMT, mast cells were enriched to homogeneity from bone marrow mononuclear cells (BM MNCs) by cell sorting for CD117+/CD34- cells. Purified mast cell populations were CD117(c-kit)+ (> 95%), CD34- (< 1%), CD3- (< 1%), CD14- (< 1%), and virtually free of contaminating cells as assessed by Giemsa staining. The genotype of MCs was analyzed after amplification by polymerase chain reaction (PCR) of a variable number tandem repeat (VNTR) region within intron 40 of the von Willebrand factor (vWF) gene. Unexpectedly, on days 88 and 126 after BMT, sorted MCs displayed recipient genotype as shown by vWF.VNTR-PCR. However, on days 198 and 494, PCR analysis showed a switch to donor genotype in isolated mast cells. Peripheral blood (PB) and BM MNC as well as highly enriched (sorted) CD3+ T cells (PB, BM), CD4+ helper T cells (PB), CD8+ T cells (PB), CD19+ B cells (PB), CD14+ monocytes (PB, BM), and CD34+ precursor cells (BM) showed donor genotype throughout the observation period. Together, these results provide evidence that human MCs developed in vivo from transplanted hematopoietic stem cells. Engraftment and in vivo differentiation of MCs from early hematopoietic progenitor cells may be a prolonged process.

Volume 193, no. 5, p. [1086–1089][1], 2011. We were informed by Kevin Young and Gang Li (Microbiology 160: 2079–2088, 2014) that strain H41 is not a tolC mutant as we had described. We have sequenced strain H41 and confirm that it is not a tolC mutant. Rather, the observed phenotype is due to a

In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli ? In a tolC mutant, 0.03 mM IPTG (isopropyl-β- d -thiogalactopyranoside) was sufficient to induce β-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce β-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum , the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli , CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.

Orthogonally protected 2,5-diaminoadipic, 2,6-diaminopimelic, and 2,7-diaminosuberic acid derivatives bearing up to four different protecting groups are prepared in one step by mixed Kolbe electrolysis. The use of 2-[(4-methylphenyl)sulfonyl]ethyl group is crucial for the isolation of the desired orthogonally protected products.

The preparation of alkyl diamino dicarboxylic acids with high optical purity (100% ee, >98.5% de) and high yields based on asymmetric catalytic hydrogenation is described. The required prochiral precursors are prepared from dialdehydes and Z-, Boc-, and acetyl-protected phosphonoglycines. Aqueous solutions of glyoxal, succinic dialdehyde, and glutaric dialdehyde were used to prepare the diunsaturated precursors for 2,5-diaminoadipic acid (DAA), 2,7-diaminosuberic acid (DAS), and 2,8-diaminoazelaic acid (DAZ). Z-Protected dimethyl esters of DAA, DAS, and DAZ were obtained by hydrogenation of the corresponding prochiral starting materials with [(COD)Rh(S,S)-Et-DuPHOS]OTf.

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.

The dynamic surface elasticity and the surface dilational viscosity of three binary phospholipid/cholesterol mixtures were determined with axisymmetric drop shape analysis on a harmonically oscillating pendent drop. Dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine were used to explore the rheological properties and phase transitions of mixtures of saturated and unsaturated phospholipids with cholesterol. The growth rates for surface dilational viscosity and dynamic elasticity are parallel for all film pressures studied. Characteristic breaks and plateaus could be found for these growth rates, indicating phase transitions. For dipalmitoylphosphatidylcholine/cholesterol and dimyristoylphosphatidylcholine/cholesterol mixtures, phase diagrams with six regions separated by phase boundaries were found, which are in good agreement with phase transitions reported in the literature for static measurements of isotherms and isobars on a Langmuir film balance and from fluorescence microscopy. Some phase boundaries were only found by dynamic, but not by static, elasticity measurements. Imaging methods revealed phase separations produced by the formation of condensed stoichiometric complexes leading to micron-sized and mostly circular domains. The effects of these complexes on monolayer rheology in liquid/liquid phases is described. Furthermore, liquid/solid and solid phase transitions are discussed.

Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P min , the bilayer lysis tensions γ l were determined. The increase in P min and γ l with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron microscopy (Cryo-TEM) images show that beside spherical, single-walled liposomes, there is a considerable fraction of discoidally deformed vesicles. Based on our results and those found in the literature, we speculate that the flattening of the vesicles is a consequence of lipid phase separation and the formation of condensed complexes and areas of different bending elasticities. This phenomenon eventually leads to agglomeration of deformed liposomal structures, becoming more pronounced with the increase in the relative amount of saturated fatty acids, presumably caused by hydrophobic interaction. For the same lipid mixture aggregation correlated linearly with the ML content. Finally, tested liposomal samples were kept at 4 °C to examine their stability. Only slight fluctuations in diameter and the increase in polydispersity after 3 weeks of storage occurred, with no statistically significant evidence of drug leakage during a time period of 12 days, illustrating physical stability of liposomes.

We study the dynamics of two phospholipid monolayers brought into contact by oblique drop impact on a liquid surface and bilayer/multilayer formation. Drop impact without monolayers shows that for low impact angles (α < 23°) and low drop velocities the drop spreads as a thin sheet on the target liquid without immersion and mixing of the two liquids. When drop and target liquid surface are covered with monolayers, bilayer/multilayer formation is expected. The composition and mechanical properties of the monolayers can strongly influence the pattern of drop impact and bilayer/multilayer formation. Monolayers with either pure saturated or unsaturated phospholipids, and their mixtures with cholesterol were used. We show that under all conditions studied bilayer/multilayer synthesis takes place. Asymmetric bilayers can be produced by the coupling of drop and target monolayers. For some lipid mixtures the drop and target monolayer collapses during drop impact and symmetric bilayers/multilayers are formed.

Mistletoe (Viscum album L.) contains the triterpene acids oleanolic acid (OA) and betulinic acid (BA), which were found to have anti-tumour properties. In this study, the solubilities of OA and BA were studied in water (up to 0.02 microg/mL in each case) and in alkaline solutions of 10 mM trisodium phosphate (pH 11.5; OA: 77.2 microg/mL; BA: 40.1 microg/mL). Furthermore, triterpene acids were quantified in aqueous mistletoe extracts (pH 7.3; drug to extract ratio 1:25). OA (1.1 microg/mL) and BA (0.9 microg/mL) were extracted with a yield of less than 5%. Preparing plant extracts with basic pH values resulted in a triterpene acid content of 9.3 microg/mL OA and 5.2 microg/mL BA (pH 12.1), reaching neither the solubility limits nor a complete extraction of the plant material. The triterpene acid content of neutral plant extracts above the solubility limit could be due to interactions with biocolloids. Interaction studies were performed by gel permeation chromatography. Different mechanisms of the dissolution at pH 7.3 and pH 10.2 are discussed.

Surface rheology of monolayers of a saturated phospholipid (dipalmitoylphosphatidylcholine, DPPC), an unsaturated phospholipid (dioleoylphosphatidylcholine, DOPC) and cholesterol is studied with axisymmetric drop shape analysis at the argon/water interface. Measurement techniques for lipids are described in detail. Profile analysis tensiometry (PAT) is used to determine the film pressure Π , surface elasticity and surface dilational viscosity of monolayers upon sinusoidal oscillations of the drop surface for various amplitudes a and frequencies f to assess their dependence on these dynamic parameters. It is shown that surface dilational viscosity strongly depends on the frequency and decreases by a factor 2–5 with increasing f in the considered range. Dilational viscosity is higher the more the monolayer approaches a relaxed state. Thus, the molecular interactions are stronger in the relaxed than in the stressed state. Surface elasticity is much less dependent on dynamic conditions. For DPPC a minimum of the dynamic surface elasticity is found for f = 12.5 mHz (at Π = 17.5 mN m −1 ) which coincides well with the relaxation frequencies measured in stress relaxation experiments. The dynamic surface elasticity of DPPC exhibits a plateau in the range 13.5 mN m −1 ≤ Π ≤ 27 mN m −1 in good coincidence with the phase boundaries of the coexistence region of micron-sized liquid crystalline domains surrounded by a fluid monolayer phase. In equilibrium measurements ( Π / A -isotherms) a plateau of the film pressure is seen at the lower bound and a break at the upper bound of the coexistence region. Film pressure/area isotherms produced by PAT and a Langmuir film balance closely coincide as is shown in a comparison to literature values. However, the surface elasticities measured dynamically with oscillating surfaces widely deviate from those derived from isotherms in the case of DPPC and cholesterol, whereas for DOPC very good agreement can be found.

The prognostic value of the detection of peripheral blood (PB) and/or bone marrow (BM) involvement by polymerase chain reaction (PCR) amplification of rearranged immunoglobulin heavy chain (IgH) and immunoglobulin kappa light chain (Igkappa) genes was evaluated in 155 patients with diffuse large B-cell lymphomas (DLBCL). Immunoglobulin gene rearrangements (IgR) were detected in 35/155 (23%) patients. The presence of IgR in PB/BM was related to clinical stage (CS I-III vs CS IV; P

The preparation of alkyl diamino dicarboxylic acids with high optical purity (100% ee, >98.5% de) and high yields based on asymmetric catalytic hydrogenation is described. The required prochiral precursors are prepared from dialdehydes and Z-, Boc-, and acetyl-protected phosphonoglycines. Aqueous solutions of glyoxal, succinic dialdehyde, and glutaric dialdehyde were used to prepare the diunsaturated precursors for 2,5-diaminoadipic acid (DAA), 2,7-diaminosuberic acid (DAS), and 2,8-diaminoazelaic acid (DAZ). Z-Protected dimethyl esters of DAA, DAS, and DAZ were obtained by hydrogenation of the corresponding prochiral starting materials with [(COD)Rh(S,S)-Et-DuPHOS]OTf.

The present study analyzes the efficiency of a combination of four immunoglobulin heavy chain (IgH) gene polymerase chain reaction (PCR) primer systems and a multiplex T-cell receptor gamma chain (TRG) gene PCR for detection of clonality in 409 samples (234 paraffin sections, 175 bone marrow aspirates) of different lymphomas. Using the four IgH PCR systems together, clonality was detected in all samples of B-cell chronic lymphocytic leukemias, hairy cell leukemias, common acute lymphoblastic leukemias, and Burkitt-like B-cell lymphomas. Clonality was detected in all bone marrow aspirates with lymphoplasmacytoid immunocytoma, mantle cell lymphoma, marginal zone B-cell lymphoma, and unclassifiable low-grade B-cell lymphomas. The combined IgH gene PCR approach allowed clonality detection in 78.2% of myelomas, 75% of Burkitt lymphomas, 74.4% of diffuse large B-cell lymphomas, 68.7% of follicular center lymphomas, 50% of posttransplant lymphomas, 28.6% of anaplastic large cell lymphomas, 29% of T-cell lymphomas, and 18.8% of Hodgkin diseases. The combination of the four IgH gene primer systems with the multiplex TRG gene PCR allowed detection of clonality in 84.2% of B-cell neoplasms, 92.1% of T-cell non-Hodgkin lymphomas, and 18.8% of Hodgkin diseases, which was much more efficient than single PCR protocols.

Chronic myeloproliferative diseases (MPDs) are not associated with consistent cytogenetic or molecular abnormalities. Demonstration of clonal cell growth by analysis of X-chromosome inactivation (XCI) patterns in females provides a promising tool for diagnosis. However, this technique can be complicated by excessive lyonization of normal cells mimicking clonal cell growth. We analyzed XCI patterns at the human androgen receptor (HUMARA) locus in 146 healthy females, 65 women with secondary neutrophilia, 31 women with reactive thrombocytosis, and 86 women with chronic MPDs. A skewed XCI pattern with greater than 75% amplification of 1 allele (allele ratio >3:1) was found in 22 (9.1%) of 242 control subjects. The incidence of skewing was statistically significantly lower in women younger than 30 years (2/73) compared with women older than 60 years (10/53). Of 86 patients with a chronic MPD, 71 (82%) exhibited an allele ratio greater than 3:1, whereas only 10 (12%) of 86 age-matched control subjects showed a skewed XCI pattern. Although statistical evaluation of the data showed a significant difference between patients with a chronic MPD and control subjects, proof of clonality in individual, especially elderly, patients is difficult.