Single-cell RNA sequencing profiling in a patient with discordant primary cutaneous B-cell and T-cell lymphoma reveals micromilieu-driven immune skewing.

BACKGROUND: Primary cutaneous lymphomas comprise a heterogeneous group of B-cell and T-cell malignancies which often show an indolent course, but can progress to aggressive disease in a subset of patients. Diagnosis is often delayed owing to clinical and histopathological similarities with benign inflammatory conditions. Especially during early disease, cancer cells are present at relatively low percentages compared with the inflammatory infiltrate, an interplay that is currently only insufficiently understood. OBJECTIVES: To improve diagnostics and perform molecular characterization of a complex type of primary cutaneous lymphoma. METHODS: Single-cell RNA sequencing (scRNA-seq) was performed and combined with T-cell and B-cell receptor sequencing. RESULTS: We were able to diagnose a patient with concurrent mycosis fungoides (MF) and primary cutaneous follicle centre lymphoma (PCFCL), appearing in mutually exclusive skin lesions. Profiling of tumour cells and the tissue microenvironment revealed a type-2 immune skewing in MF, most likely guided by the expanded clone that also harboured upregulation of numerous pro-oncogenic genes. By contrast, PCFCL lesions exhibited a more type-1 immune phenotype, consistent with its indolent behaviour. CONCLUSIONS: These data not only illustrate the diagnostic potential of scRNA-seq, but also allow the characterization of specific clonal populations that shape the unique tissue microenvironment in clinically distinct types of lymphoma skin lesions.

Changes in oestrogen receptor-alpha and -beta during progression to acquired resistance to tamoxifen and fulvestrant (Faslodex, ICI 182,780) in MCF7 human breast cancer cells.

Cell culture models of antioestrogen resistance often involve applying selective pressures of oestrogen deprivation simultaneously with addition of tamoxifen or fulvestrant (Faslodex, ICI 182,780) which makes it difficult to distinguish events in development of antioestrogen resistance from those in loss of response to oestrogen or other components. We describe here time courses of loss of antioestrogen response using either oestrogen-maintained or oestrogen-deprived MCF7 cells in which the only alteration to the culture medium was addition of 10(-6) M tamoxifen or 10(-7) M fulvestrant. In both oestrogen-maintained and oestrogen-deprived models, loss of growth response to tamoxifen was not associated with loss of response to fulvestrant. However, loss of growth response to fulvestrant was associated in both models with concomitant loss of growth response to tamoxifen. Measurement of oestrogen receptor alpha (ERalpha) and oestrogen receptor beta (ERbeta) mRNA by real-time RT-PCR together with ERalpha and ERbeta protein by Western immunoblotting revealed substantial changes to ERalpha levels but very little alteration to ERbeta levels following development of antioestrogen resistance. In oestrogen-maintained cells, tamoxifen resistance was associated with raised levels of ERalpha mRNA/protein. However by contrast, in oestrogen-deprived MCF7 cells, where oestrogen deprivation alone had already resulted in increased levels of ERalpha mRNA/protein, long-term tamoxifen exposure now reduced ERalpha levels. Whilst long-term exposure to fulvestrant reduced ERalpha mRNA/protein levels in the oestrogen-maintained cells to a level barely detectable by Western immunoblotting and non-functional in inducing gene expression (ERE-LUC reporter or pS2), in oestrogen-deprived cells the reduction was much less substantial and these cells retained an oestrogen-induction of both the ERE-LUC reporter gene and the endogenous pS2 gene which could still be inhibited by antioestrogen. This demonstrates that whilst ERalpha can be abrogated by fulvestrant and increased by tamoxifen in some circumstances, this does not always hold true and mechanisms other than alteration to ER must be involved in the development of antioestrogen resistant growth.

Hepatitis C virus among childbearing women in Scotland: prevalence, deprivation, and diagnosis.

OBJECTIVES: (A) To examine the prevalence and demographic characteristics of hepatitis C virus (HCV) infection among childbearing women in Scotland; and (B) to determine the extent of maternal HCV infection diagnosed prior to birth. METHODS: (A) Residual dried blood spot samples from routine neonatal screening, collected throughout Scotland during March-October 2000, were unlinked from identifiers and tested anonymously for HCV antibodies; and (B) electronic record linkage of Scotland's databases of births and diagnosed HCV infections was performed. RESULTS: (A) Of 30,259 samples, 121 were enzyme linked immunosorbent assay repeat reactive and 88 of these were confirmed as anti-HCV positive in the recombinant immunoblot assay, representing a seroprevalence of 0.29-0.40%. HCV seroprevalence was high among 25-29 year olds (0.4-0.57%), in high deprivation areas (0.92-1.07%), and in Greater Glasgow (0.83-0.96%) and Grampian (0.38-0.62%). Adjusted relative risk for HCV infection was highest among residents in high deprivation areas of Glasgow (7.2 (95% confidence interval 2.0-25.5)). (B) Of 121 HCV infections found among women at delivery, 24% and 46% were estimated to have been diagnosed prior to pregnancy and birth, respectively. CONCLUSIONS: HCV prevalence among Scottish childbearing women is consistent with that expected from injecting drug use. Based on reported rates of mother to child transmission, 8-11 paediatric infections are expected per annum. Diagnosis in only 24% of infected women prior to pregnancy indicates the extent to which HCV goes unrecognised in the injecting community. The current HCV screening approach-to test only those with a history of injecting drug use (or other risk factors for infection)-identifies approximately a quarter of previously undetected infections among pregnant women.

Oestrogenic activity of benzylparaben.

Previous work has demonstrated that the alkyl esters of p-hydroxybenzoic acid (parabens) possess oestrogenic activity, which increases with length of alkyl chain from methylparaben to n-butylparaben and with branching in the alkyl chain from n-butylparaben to isobutylparaben. This study reports on the oestrogenic activity of benzylparaben in a variety of assays in vitro and in vivo. Benzylparaben was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor (ER) of MCF7 human breast cancer cells by 22% at 1000-fold molar excess, by 40% at 10,000-fold molar excess, by 57% at 100 000-fold molar excess and by 100% at 1,000,000-fold molar excess. It was able to increase expression of a stably transfected oestrogen responsive reporter gene (ERE-CAT) in MCF7 cells after 24 h at 10(-5)M/10(-4)M and after 7 days at 10(-6)M/10(-5)M/10(-4)M. Proliferation of MCF7 cells could be increased by 10(-6)M/10(-5)M benzylparaben and this could be inhibited by 10(-7)M pure anti-oestrogen ICI 182,780, indicating that growth effects were ER mediated. Further evidence for ER-mediation was provided from the ability of benzylparaben to increase the growth of a second oestrogen-dependent human breast cancer cell line ZR-75-1, but not the oestrogen-insensitive MDA-MB-231 cell line. When tested in the presence of 10(-10)M 17beta-oestradiol, benzylparaben gave no antagonist response on the growth of either MCF7 or ZR-75-1 cells. Finally, benzylparaben could increase uterine weight in the immature mouse following topical application of three daily doses of 33 mg to dorsal skin. These results demonstrate that the oestrogenicity of methylparaben can be increased by the addition of an aryl group as well as by lengthening or branching the alkyl grouping.

Oestrogenic activity of isobutylparaben in vitro and in vivo.

The alkyl esters of p-hydroxybenzoic acid (parabens) are used widely as preservatives in foods, pharmaceuticals and cosmetics to which the human population is exposed. Recent studies have reported that methylparaben, ethylparaben, n-propylparaben and n-butylparaben all possess oestrogenic activity in several in vitro assays and in animal models in vivo. This study reports on the oestrogenic activity of isobutylparaben in a panel of assays in vitro and in vivo. Isobutylparaben was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor alpha of MCF7 human breast cancer cells by 81% at 100 000-fold molar excess. Using a clonal line of MCF7 cells containing a stably transfected oestrogen-responsive ERE-CAT reporter gene, CAT gene expression could be increased by isobutylparaben such that the magnitude of the response was the same at 10(-5) M isobutylparaben as with 10(-8) M 17beta-oestradiol. Isobutylparaben could also increase expression of the endogenous oestrogen-responsive pS2 gene in MCF7 cells and maximal expression at 10(-5) M isobutylparaben could be inhibited with the anti-oestrogen ICI 182 780. The proliferation of two oestrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1 could be increased with isobutylparaben such that at concentrations of 10(-5) M the proliferation response was of the same magnitude as with 10(-8) M 17beta-oestradiol. Evidence for oestrogen receptor mediation of proliferation effects was provided by the inability of isobutylparaben to influence the growth of oestrogen-unresponsive MDA-MB-231 human breast cancer cells and by the ability of the anti-oestrogen ICI 182 780 to inhibit the isobutylparaben effects on MCF7 cell growth. The proliferation response to 10(-10) M 17beta-oestradiol was not antagonized with isobutylparaben at any concentration from 10(-9) M to 10(-4) M in either MCF7 or ZR-75-1 cells. Finally, subcutaneous administration of isobutylparaben was able to increase the uterine weight in the immature mouse after three daily doses of 1.2 or 12.0 mg per mouse. Previous work using linear-alkyl-chain parabens has shown that oestrogenic activity increases with alkyl chain length from methylparaben to n-butylparaben. The results here show that branching of the alkyl chain to isobutylparaben increases oestrogenic activity beyond that of the equivalent length linear alkyl chain in n-butylparaben.

Oestrogenic activity of parabens in MCF7 human breast cancer cells.

Parabens (4-hydroxybenzoic acid esters) have been recently reported to have oestrogenic activity in yeast cells and animal models. Since the human population is exposed to parabens through their widespread use as preservatives in foods, pharmaceuticals and cosmetics, we have investigated here whether oestrogenic activity of these compounds can also be detected in oestrogen-sensitive human cells. We report on the oestrogenic effects of four parabens (methylparaben, ethylparaben, n-propylparaben, n-butylparaben) in oestrogen-dependent MCF7 human breast cancer cells. Competitive inhibition of [3H]oestradiol binding to MCF7 cell oestrogen receptors could be detected at 1,000,000-fold molar excess of n-butylparaben (86%), n-propylparaben (77%), ethyl-paraben (54%) and methylparaben (21%). At concentrations of 10(-6)M and above, parabens were are able to increase expression of both transfected (ERE-CAT reporter gene) and endogenous (pS2) oestrogen-regulated genes in these cells. They could also increase proliferation of the cells in monolayer culture, which could be inhibited by the antiestrogen ICI 182,780, indicating that the effects were mediated through the oestrogen receptor. However, no antagonist activity of parabens could be detected on regulation of cell proliferation by 17 beta-oestradiol at 10(-10)M. Molecular modelling has indicated the mode by which paraben molecules can bind into the ligand binding pocket of the crystal structure of the ligand binding domain (LBD) of the oestrogen receptor alpha (ERalpha) in place of 17beta-oestradiol; it has furthermore shown that two paraben molecules can bind simultaneously in a mode in which their phenolic hydroxyl groups bind similarly to those of the meso-hexoestrol molecule. Future work will need to address the extent to which parabens can accumulate in hormonally sensitive tissues and also the extent to which their weak oestrogenic activity can add to the more general environmental oestrogen problem.

Insulin-like growth factor receptor levels are regulated by cell density and by long term estrogen deprivation in MCF7 human breast cancer cells.

This work describes a reciprocal relationship between cell density and levels of insulin-like growth factor receptors (IGFR) in MCF7 human breast cancer cells, which adds a new dimension to the mechanism of cross-talk between estrogen and insulin-like growth factors in the regulation of breast cancer cell growth. The reduced binding of both (125)I-IGF1 and alphaIR3 anti-IGFR antibody to whole cells showed that IGFR are lost from the surface of MCF7 cells as cell density increases, and this occurred irrespective of the presence or absence of estradiol. Western immunoblotting further confirmed loss of type I IGFR from MCF7 cells with increasing cell density. Long term estrogen deprivation was found to increase the levels of IGFR at all cell densities, such that after 96 weeks of estrogen deprivation, IGFR levels had become similar at the highest cell density in the absence of estradiol to the IGFR levels at the lowest cell density in the estrogen-maintained cells, and the levels of IGFR could be increased still further by estradiol. This overexpression of IGFR in the estrogen-deprived cells correlated with a reversal of response to exogenously added ligand, in that concentrations of insulin, IGFI, and IGFII that had stimulated growth of the estrogen-maintained cells became growth inhibitory to the estrogen-deprived cells. Blockade of the IGFIR with the alphaIR3 anti-IGFR antibody could partially inhibit the growth of the estrogen-deprived cells, suggesting that up-regulation of IGFR in these cells may contribute to the mechanism of adaptation to growth in steroid-deprived conditions which results in progression to estrogen independence of cell growth.

Wavelength-dependent photochemistry in Cr(CNPh)6: a study of photosubstitution and photoinduced electron transfer using time-resolved spectroscopy.

The wavelength dependence of photosubstitution, photoinduced electron transfer, and the time-resolved spectra of Cr(CNPh)6, a compound having low-lying MLCT states, were investigated. Photosubstitution quantum yields increase with increasing excitation energy while photoinduced electron transfer quantum yields decrease with increasing excitation energy. At the lowest excitation energy used (532 nm, or 18,800 cm(-1)), the quantum yields for both electron transfer and photosubstitution reach the same maximum value, 0.29. Picosecond time-resolved absorption spectra at 355 and 532 nm excitation wavelengths show two features: a bleach signal centered at 400 nm and an excited state absorption (ESA) in the 600 nm region. The ESA signal is much weaker for 532 nm excitations than for 355 nm excitations. Following a 355 nm flash, the bleach and ESA decay exponentially with the same lifetime of 23 micros. This implies a simple ligand dissociation followed by recombination. Bleach recovery kinetics after a 532 nm flash are more complicated: two or three exponential components are required to fit the data. Cr(CNPh)6 exhibits two photochemical mechanisms: at high excitation energy, a simple charge neutral dissociation occurs; at low energy, it is proposed that a phenylisocyanide radical anion dissociates, forming a radical pair that is responsible for the observed substitution and electron transfer reactivity, and the complicated nanosecond kinetics. The primary processes for both reactions occur in less than 20 ps.

Two-week dietary soy supplementation has an estrogenic effect on normal premenopausal breast.

An association has been reported between consumption of a high soy diet and a low incidence of breast cancer within populations of Southeast Asia. Phytoestrogens present in soy act as partial estrogen agonists or antagonists and can inhibit breast cancer cell proliferation in vitro. The effect of 14-day dietary soy supplementation with 60 g (45 mg isoflavones) on the normal breast of 84 premenopausal patients was determined. Serum concentrations of the isoflavanoids, genistein, daidzein, and equol, were raised in patients after soy supplementation (P < or = 0.025). Nipple aspirate (NA) levels of genistein and daidzein were higher than paired serum levels, both before (P < 0.001 and P = 0.001, respectively) and after soy supplementation (P < 0.001 and P = 0.049, respectively); however, there was no significant increase in NA isoflavone levels in response to soy. NA levels of apolipoprotein D were significantly lowered and pS2 levels raised in response to soy supplementation (P < or = 0.002), indicative of an estrogenic stimulus. No effect of soy supplementation on breast epithelial cell proliferation, estrogen and progesterone receptor status, apoptosis, mitosis, or Bcl-2 expression was detected. In conclusion, short term dietary soy has a weak estrogenic response on the breast, as measured by nipple aspirate apolipoprotein D and pS2 expression. No antiestrogenic effect of soy on the breast was detected.

mucK, a gene in Acinetobacter calcoaceticus ADP1 (BD413), encodes the ability to grow on exogenous cis,cis-muconate as the sole carbon source.

Benzyl alcohol, benzaldehyde, benzoate, and anthranilate are metabolized via catechol, cis,cis-muconate, and the beta-ketoadipate pathway in Acinetobacter calcoaceticus ADP1 (BD413). Mutant strain ISA25 with a deletion spanning catBCIJF and unable to metabolize muconate further will not grow in the presence of an aromatic precursor of muconate. Growth on fumarate as the sole carbon source with added benzyl alcohol or benzaldehyde selected spontaneous mutants of ISA25. After repair of the cat deletion by natural transformation with linearized plasmid pPAN4 (catBCIJF) 10 mutants were unable to grow on benzoate of cis,cis-muconate but could still grow on anthranilate. Transformation with wild-type chromosomal DNA demonstrated the presence of two unlinked mutations in each strain, one in the benABCD region, encoding the conversion of benzoate to catechol, and the other in a gene determining the ability to grow on exogenous cis,cis-muconate. The wild-type gene, named mucK, was cloned into pUC18, and its nucleotide sequence was determined. It encodes a 413-residue protein of M(r) = 45,252 which is a member of a superfamily of membrane transport proteins and which is within a subgroup involved in the uptake of organic acids. Five of the mutant alleles were cloned, and the mutations were determined by nucleotide sequencing. All the mutations were in the mucK coding region and consisted of three deletions, one duplication, and a substitution. Insertional inactivation of mucK resulted in the loss of the ability to utilize exogenous muconate. The location of mucK on the chromosome appeared to be unique for genes associated with the benzoate branch of the beta-ketoadipate pathway in being close to the pca-qui-pob gene cluster (for p-hydroxybenzoate utilization) and distant from the functionally related ben-cat cluster. Downstream of mucK and transcribed in the same direction is an open reading frame encoding a protein of 570 residues (M(r) = 63,002) which shows considerable homology with a mammalian electron transport protein; its insertional inactivation had no detectable phenotypic effect.

Two identical copies of IS1246, a 1275 base pair sequence related to other bacterial insertion sequences, enclose the xyl genes on TOL plasmid pWW0.

Two identical direct repeats of a 1275 bp sequence, designated IS1246, encompass the xyl genes, which determine the catabolism of toluene, m- and p-xylenes to central metabolites, on the TOL catabolic plasmid pWW0. IS1246 has a terminal inverted repeat of 12 bp (5'GGGCACCTCGAA3') and contains a major open reading frame of 280 codons. This ORF shows significant homology with ORFs encoded by a number of bacterial insertion sequences from Bacteroides, Neisseria and Escherichia coli.

The lower pathway operon for benzoate catabolism in biphenyl-utilizing Pseudomonas sp. strain IC and the nucleotide sequence of the bphE gene for catechol 2,3-dioxygenase.

Pseudomonas sp. strain IC is able to grow on biphenyl, 3-methylbiphenyl and 4-methylbiphenyl. These are converted to benzoate and the corresponding methylbenzoates. The lower pathway genes for the catabolism of the benzoates were cloned on a 22 kb HindIII fragment. Hybridization with gene-specific probes from the meta pathways of other catabolic plasmids showed that the gene order was identical to that of the operons carrying the same function from TOL plasmids. The nucleotide sequence of a 1241 bp region carrying the whole of the bphE gene (for a catechol 2,3-dioxygenase) and the 5' end of the downstream bphG gene (for 2-hydroxy semialdehyde dehydrogenase) was determined. Both genes showed a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas strains. The upper pathway genes for the conversion of biphenyl to benzoate have also been cloned but no linkage with the lower pathway operon has been detected. Pseudomonas strain IC contains a large plasmid pWW110 (> 200 kb) and there are indications that this plasmid carries the bph genes.

A comparison of the multiple alleles of xylS carried by TOL plasmids pWW53 and pDK1 and its implications for their evolutionary relationship.

Both of the independently isolated TOL plasmids pWW53 and pDK1 contain multiple regions homologous to the xylS regulatory gene of the archetypal TOL plasmid pWW0. The three homologues on pWW53 vary in the extent of their homology to xylSpWW0, xylS1pWW53 is 99% identical to xylSpWW0 and is located relative to the single copy of xylRpWW53 in exactly the same way as xylS and xylR on pWW0. The DNA sequence of xylS3pWW53 is 87% identical to the xylSpWW0 sequence within the coding region but the non-coding DNA upstream is not homologous. There is a frame-shift change at the end of the coding region which causes the C terminus of XylS3pWW53 to be extended by an additional 10 amino acids relative to XylSpWW0. xylS2pWW53 is anomalous and appears to encode a truncated pseudogene lacking the first 525 bases found in the other xylS genes. Evidence is presented to show that both xylS1pWW53 and xylS3pWW53 act as regulators of meta pathway operons. Plasmid pDK1 carries two homologues of xylS. xylS1pDK1 is functional and is a hybrid gene: its 5' end and the upstream sequences are highly homologous to both xylS1pWW53 and xylSpWW0, whereas its 3' end is identical to xylS3pWW53. The sequence of xylS2pDK1 is identical to that of the anomalous truncated xylS2pWW53. Comparison of the organization and the restriction maps of the xyl catabolic operons on pDK1 and pWW53, together with the nucleotide sequences presented here, indicates that the catabolic DNA on pDK1 has derived from a replicon on which the xyl genes are organized similarly to pWW53 and that a genetic rearrangement has taken place involving a reciprocal recombination internal to two of its xylS homologues.

Construction of hybrid xylE genes between the two duplicate homologous genes from TOL plasmid pWW53: comparison of the kinetic properties of the gene products.

The two xylE genes for catechol 2,3-oxygenase, encoded by TOL plasmid pWW53, carry a common SalI restriction site within the reading frame. Each gene was cut at the SalI site and the 5' end of each gene spliced to the 3' end of the other to form hybrid genes, from both of which catalytically active catechol 2,3-oxygenase activities were expressed. The kinetic parameters were determined for the gene products of both the hybrid and the wild-type xylE genes with catechol, 3-methylcatechol and 4-methylcatechol as substrates. Comparison of the results suggested firstly, that the C-terminal regions of the enzymes determined both the binding and the catalytic specificity, and, secondly, that the N-terminal region of one of the enzymic gene products contained a secondary binding site which caused inhibition by excess substrate for methylcatechol substrates but not for catechol. One of the wild-type enzymes appeared to have an intrinsically higher activity for all three substrates than the other. This higher activity depended on the presence of both its C- and N-terminal regions, and in both hybrid enzymes, which contained only one of these regions, activity was significantly reduced.

Physical and functional mapping of two cointegrate plasmids derived from RP4 and TOL plasmid pDK1.

Cointegrate plasmids were formed in vivo between the broad-host-range R-plasmid RP4 and two catabolic plasmids derived from Pseudomonas putida HS1. One of these was the wild-type plasmid pDK1 encoding the complete inducible toluene/xylene (TOL) catabolic pathway and one was pDKT1, a deletion derivative of pDK1 selected after growth of HS1 on benzoate and supporting growth on only toluene. The two plasmids formed, pDK2 and pDKT2 respectively, each consisted of a complete RP4 replicon in which was an insert of the parent plasmid DNA respectively 40 and 20 kbp in size. The detailed restriction maps of the two plasmids were determined and many of the catabolic genes were located by subcloning and enzyme assay of recombinant plasmids in Escherichia coli and Pseudomonas hosts. The insert in pDK2 contained both operons of the catabolic pathway, the 'upper pathway' operon (xylCAB) and the meta pathway operon (xylDLEGF(I,J,K)H), and a region identified as having the function of the regulator gene xylS. The insert in pDKT2 contained only the upper pathway operon and the regulatory region. Within each of the three coding regions there was great similarity with the same regions on TOL plasmids pWW0 and pWW53-4 apparent (a) by the same order of the genes, (b) by a similar pattern of restriction sites and (c) by hybridization studies. However, the order and orientations of the three coding regions differed from those previously described for both pWW0 and pWW53-4. The significance of these findings to the evolution of TOL plasmids is discussed.