Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.     

Culture of tumour-infiltrating lymphocytes from melanoma and colon carcinoma: Removal of tumour cells does not affect tumour-specificity

The therapeutic potential of adoptive therapy using tumour-infiltrating lymphocytes (TIL) has been demonstrated in a number of clinical trials. However, freshly isolated tumour-infiltrating lymphocytes (TIL) are often impaired in their proliferative and cytotoxic responses, which limits their use in immunotherapy. Several hypotheses with regard to the poor effector function of TIL have been postulated, including the production of immunosuppressive factors by tumour cells. In a previous paper we reported the efficient expansion of immunoreactive TIL from a variety of solid tumours by stimulation with a combination of monoclonal antibodies (mAbs) against CD3 and CD28. In the present study we analysed whether this protocol would be improved by the removal of tumour cells at the start of the culture. We tested a highly immunogenic tumour, melanoma, and a poorly immunogenic tumour, colon carcinoma. Removal of tumour cells highly improved anti-CD3/CD28 stimulated expansion of TIL from colon carcinoma, resulting in a significantly higher percentage of potentially tumour-specific CD8-positive T-cells and a reduced CD4/CD8 ratio compared to expansion in the presence of tumour cells. In contrast, expansion and CD4/CD8 ratio of melanoma-derived TIL was not significantly influenced by the removal of autologous tumour cells. CD3/CD28-stimulated melanoma TIL cultured in the absence of tumour cells showed specific lysis of autologous tumour cells comparable to melanoma TIL cultured in high-dose IL2. However, no cytotoxicity could be detected in colon TIL irrespective of the culture conditions used. On the other hand, 3/8 colon carcinoma TIL cultures and 9/12 melanoma-derived TIL cultures showed IFN secretion upon stimulation with autologous tumour cells. We conclude that stimulation of TIL with a combination of mAbs to CD3 and CD28 in the absence of tumour cells induces efficient expansion of potentially tumour-specific cells from a highly and a poorly immunogenic tumour. Removal of tumour cells does not have a negative influence on the generation of tumour-specific T cells, while cell yield improves. Therefore, for large-scale cultures this protocol can efficiently induce the outgrowth of tumour-specific TIL, at the same time providing a useful source of autologous tumour cells that can be stored and used to direct or test antitumour specificity.     

Complementation experiments between conditional lethal mutants of bacteriophage ?X174

Summary Complementation experiments with temperature sensitive (ts) and suppressor sensitive (sus) mutants of bacteriophage X174 unambiguously revealed five cistrons on the basis of a clear bipartition of burst sizes.A new group of sus mutants (emeralds) was found, defective in a function essential for growth in Shigella sonnei V64.The complementation between ts and sus mutants was in general asymmetric in that the yield of ts particles was lower than that of the sus particles. The mutants of one cistron (defective in RF-replication) showed a completely asymmetric complementation behaviour both of ts and sus mutants. The ts mutants of this group, which show to be early, appear to be defective in two functions.The possibility is discussed that in each cell only one phage genome is replicated. This would explain both kinds of asymmetric complementation and the low burst sizes that were obtained when mutants of particular genes were complemented.     

Reduced expression of distinct T-cell CD molecules by collagenase/DNase treatment

DNase/collagenase treatments are widely used to obtain single-cell suspensions of tumour cells and tumour-infiltrating T lymphocytes (TIL) from solid tumours. Since the functional integrity of such cells has been questioned, we have studied whether treatments with commonly used preparations of these enzymes could affect the expression of lymphocyte surface molecules and lymphocyte proliferative responsiveness. With peripheral-blood-derived T cells as a model, flow-cytometric analysis revealed strongly reduced expression of distinct CD molecules for each enzyme, notably CD2, CD4, CD8 and CD44 for DNase, and CD4, CD14, CD16, and CD56 for collagenase. The effects were found to be due to protease contaminations present in all but the purest enzyme preparations tested. Addition of serum or trypsin inhibitor abolished the effects. Since serum-free media are widely used to expand tumour-infiltrating T cells for clinical therapeutic use, data from early phenotypic analyses can be strongly misleading. Even after an 18-h rest period following the enzyme treatments, re-expression of the affected membrane markers was still far from complete. On the other hand, despite strongly reduced expression of CD2 molecules on the lymphocyte membrane, anti-CD2-induced proliferation was not affected, showing the redundancy of this signal molecule. Since other important T cell activation molecules (TCR, CD3, CD28) were not affected by enzymatic treatment, the use of expensive, highly purified collagenase/DNase preparations does not seem to be mandatory in clinical studies with expanded TIL.     

Structure-activity analysis of peptidic Chlamydia HtrA inhibitors

Chlamydia trachomatis high temperature requirement A (CtHtrA) is a serine protease that performs proteolytic and chaperone functions in pathogenic Chlamydiae; and is seen as a prospective drug target. This study details the strategies employed in optimizing the irreversible CtHtrA inhibitor JO146 [Boc-Val-Pro-Val^P(OPh)₂] for potency and selectivity. A series of adaptations both at the warhead and specificity residues P₁ and P₃ yielded 23 analogues, which were tested in human neutrophil elastase (HNE) and CtHtrA enzyme assays as well as Chlamydia cell culture assays. Trypsin and chymotrypsin inhibition assays were also conducted to measure off-target selectivity. Replacing the phosphonate moiety with α-ketobenzothiazole produced a reversible analogue with considerable CtHtrA inhibition and cell culture activity. Tertiary leucine at P₃ (8a) yielded approximately 33-fold increase in CtHtrA inhibitory activity, with an IC₅₀ = 0.68 ± 0.02 µM against HNE, while valine at P₁ retained the best anti-chlamydial activity. This study provides a pathway for obtaining clinically relevant inhibitors.     

The temperature activated HtrA protease from pathogen Chlamydia trachomatis acts as both a chaperone and protease at 37 degrees C

Characterization of the protease, HtrA, from pathogen Chlamydia trachomatis is presented. The purified recombinant protein was a serine endoprotease, specific for unfolded proteins, and temperature activated above 34 degrees C. Chaperone activity was observed, although this appeared target-dependent. Inactive protease (S247A) was able to chaperone insulin B-chain, irrespective of temperature, but at 30 degrees C only HtrA and not S247A displayed significant chaperone activity for alpha-lactalbumin. These data demonstrate that chaperone activity may involve functional protease domain and that C. trachomatis HtrA functions as both a chaperone and protease at 37 degrees C. These properties are consistent with the developmental cycle of this obligate intracellular bacterium.     

Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

Bacteriophages P2, P2 Hy dis and WΦ are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein–DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein–DNA complexes, and only WΦ was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WΦ operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WΦ C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages.     

Absence of differentiation-related expression of keratin 10 in earlystages of vulvar squamous carcinoma

Using specific monoclonal antibodies (DE-K10 and DE-SCK respectively), the expression of some differentiation-related epidermal keratins was studied in 38 human vulvar squamous carcinomas. In the epidermis, expression of keratin 10 (K10) strictly paralleled the extent of differentiation; it was absent in the basal layer, appeared in the first suprabasal layers and increased in concentration towards the granular layer. However, K10 was rarely detected (1 case out of 12) in early stages of vulvar squamous carcinomas (tumours less than 2 cm, clinical stage I) regardless of the tumour grade. In larger and more advanced tumours (greater than 2 cm, clinical stages II and III), K10 was detected in 21 out of 26 cases. Its expression appeared to be related to maturation of malignant keratinocytes, being preferentially detected in more-differentiated parts. Occasionally however, cells that did not show histological signs of keratinisation were also K10-positive. Modified stratum corneum keratins (recognized specifically by monoclonal antibody DE-SCK) were detected in the most keratinized areas (horn pearls and their close vicinity) of some K10-positive tumours, i.e., in a pattern close to their normal expression in terminally differentiated epidermal cells. These data suggest differences in the regulation of K10 expression during the differentiation processes in the normal keratinising squamous epithelium and in squamous carcinomas. While the normal pattern of vulvar epithelial differentiation is accompanied by an increasing expression of K10, malignant keratinocytes, also when these are histologically moderately or well differentiated, cease expressing this keratin in the early stages of tumour development.