ABSTRACT
BACKGROUND: First- and third-generation retinoids are the main treatment for acne. Even though efficacious, they lack full selectivity for retinoic acid receptor (RAR) γ, expressed in the epidermis and infundibulum. OBJECTIVES: To characterize the in vitro metabolism and the pharmacology of the novel retinoid trifarotene. MATERIALS AND METHODS: In vitro assays determined efficacy, potency and selectivity on RARs, as well as the activity on the expression of retinoid target genes in human keratinocytes and ex vivo cultured skin. In vivo studies investigated topical comedolytic, anti-inflammatory and depigmenting properties. The trifarotene-induced gene expression profile was investigated in nonlesional skin of patients with acne and compared with ex vivo and in vivo models. Finally, the metabolic stability in human keratinocytes and hepatic microsomes was established. RESULTS: Trifarotene is a selective RARγ agonist with > 20-fold selectivity over RARα and RARß. Trifarotene is active and stable in keratinocytes but rapidly metabolized by human hepatic microsomes, predicting improved safety. In vivo, trifarotene 0·01% applied topically is highly comedolytic and has anti-inflammatory and antipigmenting properties. Gene expression studies indicated potent activation of known retinoid-modulated processes (epidermal differentiation, proliferation, stress response, retinoic acid metabolism) and novel pathways (proteolysis, transport/skin hydration, cell adhesion) in ex vivo and in vivo models, as well as in human skin after 4 weeks of topical application of trifarotene 0·005% cream. CONCLUSIONS: Based on its RARγ selectivity, rapid degradation in human hepatic microsomes and pharmacological properties including potent modulation of epidermal processes, topical treatment with trifarotene could result in good efficacy and may present a favourable safety profile in acne and ichthyotic disorders.
Subject(s)
Acne Vulgaris/drug therapy , Dermatologic Agents/pharmacology , Receptors, Retinoic Acid/agonists , Retinoids/pharmacology , Acne Vulgaris/pathology , Administration, Cutaneous , Animals , Biopsy , Cell Differentiation/drug effects , Cell Line , Dermatologic Agents/therapeutic use , Disease Models, Animal , Drug Evaluation, Preclinical , Drug Stability , Gene Expression/drug effects , Gene Expression Profiling , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice , Microsomes, Liver , Retinoids/therapeutic use , Skin , Skin Pigmentation/drug effects , Tissue Culture Techniques , Retinoic Acid Receptor gammaABSTRACT
BACKGROUND: Possible outcomes of acne lesions are atrophic scars, which may cause serious psychological distress. Current treatments for postacne scarring often require invasive procedures. Pathophysiological studies on acne scarring have only investigated the first week of papule life. OBJECTIVES: To study the pathophysiology of atrophic scar formation to identify molecular and cellular pathways that can lead to new therapies for the prevention of acne scarring. METHODS: Large-scale gene expression profiling and immunohistochemistry analysis were performed on uninvolved skin and papules in both scar-prone (SP) and non-scar-prone (NSP) patients with acne, at different time points. RESULTS: Gene expression and immunohistochemistry analyses showed a very similar immune response in 48-h-old papules in SP and NSP populations, characterized by elevated numbers of T cells, neutrophils and macrophages. However, the immune response only persisted in SP patients in 3-week-old papules, and was characterized by an important B-cell infiltrate. Transient downmodulation of sebaceous gland markers related to lipid metabolism was observed in 48-h-old papules in NSP patients, followed by normalization after 3 weeks. In contrast, in SP patients a drastic reduction of these markers persisted in 3-week-old papules, suggesting an irreversible destruction of sebaceous gland structures after inflammatory remodelling in SP patients with acne. CONCLUSIONS: Long-lived acne papules are characterized by a B-cell infiltrate. A relationship exists between the duration and severity of inflammation and the alteration of sebaceous gland structures, leading to atrophic scar formation in acne.
Subject(s)
Acne Vulgaris/complications , Cicatrix/immunology , Plasma Cells/immunology , Sebaceous Glands/pathology , Atrophy/etiology , Atrophy/immunology , Biopsy , Cicatrix/etiology , Cicatrix/pathology , Epidermis/immunology , Epidermis/pathology , Gene Expression Profiling , Humans , Sebaceous Glands/cytology , Sebaceous Glands/immunologyABSTRACT
The general properties of the taurine uptake in human endometrial tumoral Ishikawa cells were similar to those usually found in other tissues. Uptake was notably affected by the oxygen pressure, being higher at the physiological pO(2) of the endometrium (40 mm Hg, equivalent to 5% O(2)) compared to that used under standard experimental culture conditions (160 mm Hg or 20% O(2)). Uptake of taurine was also density-dependent in Ishikawa cells and was significantly decreased at confluence. Uptake regulation by PKC driven phosphorylation occurs only in growing cells and not in resting cells. The taurine uptake of three Ishikawa cell lines was very different. The taurine uptake of one of the cell lines was affected by estradiol, probably through a non-genomic pathway, whereas tamoxifen had no effect in all cell lines.
Subject(s)
Endometrial Neoplasms/metabolism , Taurine/pharmacokinetics , Cell Count , Cell Division/drug effects , Cell Line, Tumor , Culture Media/pharmacology , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/pathology , Estradiol/pharmacology , Female , Humans , Oxygen/pharmacology , Phenolsulfonphthalein/pharmacology , Tamoxifen/pharmacology , Taurine/antagonists & inhibitors , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Fifty-seven cases of Ig light chain-associated Fanconi syndrome (FS) have been reported so far, mostly as isolated reports. The pioneering work by Maldonado and associates (35), who reviewed the first 17 cases in 1975, led to the unifying concept that patients with FS and Bence Jones proteinuria have a special form of plasma cell dyscrasia characterized by slow progression of the tumor and by prominent crystal formation in proximal tubule cells, in the absence of myeloma casts in the distal tubule. We carefully reappraised these characteristics in a series of 11 patients. Ten renal biopsy specimens were available for electron microscopy, adding to the 15 previously reported cases with ultrastructural studies. Moreover, 10 of the kappa light chains could be entirely or partially sequenced and tested for their resistance to cathepsin B, a lysosomal protease present in proximal tubule cells. Our series showed an unexpected clinicopathologic heterogeneity. Seven patients presented with the typical clinical and pathologic features of FS and low-mass myeloma or monoclonal gammopathy of undetermined significance (MGUS), in keeping with Maldonado et al's description. Crystals in bone marrow cells were detected in patients of this group, only. Three patients who presented with full-blown FS exhibited, however, the characteristic features of myeloma cast nephropathy in the setting of high-mass myeloma. One patient of this group also had numerous crystals in proximal tubule cells. The eleventh patient had complete FS with MGUS, but no crystals in proximal tubule cells even after electron microscopy. Contrasting with the clinicopathologic heterogeneity, genetic and biochemical analyses of the light chains showed a striking homogeneity. First, they all were of the kappa type. Second, 8 of 9 belonged to the V kappa I variability subgroup, which indicates that FS light chains are related by the sequence of their variable regions. Third, the 8 V kappa I light chain sequences most likely originated from only 2 germline genes, LCO2/012 and LCO8/018. Fourth, all 5 LCO2/012-derived sequences presented an unusual hydrophobic or nonpolar residue at position 30. These sequence peculiarities may account for unusual physicochemical properties of the light chains including the resistance of their variable domain V kappa to proteolysis by cathepsin B, observed in 7 of 9 patients in our series, while light chains isolated from patients with myeloma cast nephropathy are completely digested. Resistance of V kappa to proteolysis in FS patients can explain the accumulation of the light chain in the endocytotic compartment of the proximal tubule cells, leading to impairment of proximal tubule functions.
Subject(s)
Fanconi Syndrome/immunology , Paraproteinemias/complications , Adult , Aged , Aged, 80 and over , Fanconi Syndrome/mortality , Fanconi Syndrome/pathology , Female , Humans , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/urine , Immunoglobulin kappa-Chains/chemistry , Immunoglobulin kappa-Chains/urine , Kidney Tubules, Proximal/pathology , Male , Middle Aged , Multiple Myeloma/etiology , Multiple Myeloma/immunology , Paraproteinemias/immunology , Paraproteinemias/pathologyABSTRACT
Crystal formation by monoclonal immunoglobulins is a well-known but rare complication of B-cell neoplasia. We have designed an in vivo model of cryocrystalglobulinemia by grafting to mice hybridoma clones producing a pathogenic monoclonal immunogloblulin (Ig) G3kappa. One clone, 8A4, secreted a singular IgG3 that formed crystals both in the proliferating plasma cells and as mesangial and subendothelial deposits in the kidney glomeruli. Morphologic analysis of kidneys revealed neutrophil infiltration and endocapillary hyperplasia, while the morphology of deposits was reminiscent of those in cryocrystalglobulinemia patients. A variant clone that only differed from 8A4 by a 3-amino acid deletion in the V(kappa) CDR1 increased its secretion level by 7-fold and produced an abundant bona fide serum monoclonal cryoglobulin in mice, without crystal formation within tumoral cells; it yielded no subendothelial deposits but only amorphous precipitates in capillary lumens of kidney glomeruli, reminiscent of those seen in the human hyperviscosity syndrome, without other glomerular lesions. A limited variation in the V(kappa) domain thus proved able to increase secretion, to abrogate crystallization, and to modify patterns of glomerular lesions and deposits. Both the crystallizing and noncrystallizing IgG3kappa sequences were related to previously reported murine cryoglobulins, all including a gamma3 chain and canonical VH sequences. Two additional variants of 8A4 with identical VH and VL domains but having switched to IgG1 also lost crystal formation, further showing this feature of 8A4 to result from a unique 3-dimensional conformation of the complete immunoglobulin, relying on V and C domain primary structures of both chains.
Subject(s)
Cryoglobulinemia/immunology , Cryoglobulinemia/pathology , Immunoglobulin G/immunology , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/immunology , Immunoglobulin Variable Region/immunology , Kidney Glomerulus/immunology , Kidney Neoplasms/immunology , Kidney/immunology , Amino Acid Sequence , Animals , Capillaries/pathology , Cryoglobulins/analysis , Disease Models, Animal , Female , Humans , Hybridomas , Hyperplasia , Immunoglobulin G/chemistry , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Light Chains/chemistry , Immunoglobulin Variable Region/chemistry , Kidney/blood supply , Kidney/pathology , Kidney Glomerulus/pathology , Kidney Glomerulus/ultrastructure , Kidney Neoplasms/pathology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neutrophils/pathologyABSTRACT
Plasma cell dyscrasias may be responsible for Fanconi's syndrome, due to the toxicity of a free monoclonal kappa light chain toward kidney proximal tubules. Eight cases of Fanconi's syndrome were analyzed. We compared the structures of VkappaI variability subgroup V domains from five cases of Fanconi's syndrome and one myeloma without renal involvement. Among Fanconi cases, four putative structures were obtained after molecular modeling by homology, and the other had previously been refined by X-ray crystallography. The complete sequences of one VkappaI, one VkappaIII and N-terminal sequences of two VkappaI light chains, from patients with different forms of Fanconi's syndrome, were compared with four previously studied sequences. All three kappa chains responsible for a 'classical' form with intralysosomal crystals and a low mass myeloma, were encoded by the LCO2/O12 germline gene and had an unusual non-polar residue exposed to the solvent in the CDR-L1 loop. Of both VkappaI light chains from patients with Fanconi's syndrome without intracellular crystals, one derived from LCO2/O12 and the other from LCO8/O18 gene. Another feature that could be related to non-crystallization was the absence of accessible side chains in the CDR-L3 loop which is known to be implicated in dimer formation.
Subject(s)
Antibodies, Monoclonal/chemistry , Fanconi Syndrome/immunology , Immunoglobulin Light Chains/chemistry , Immunoglobulin kappa-Chains/chemistry , Amino Acid Sequence , Cathepsin B/pharmacology , Electrophoresis, Polyacrylamide Gel , Humans , Models, Molecular , Molecular Sequence Data , Peptide Hydrolases/pharmacology , Sequence Analysis , Sequence Homology, Amino AcidABSTRACT
Monoclonal free light chains secreted in immunoproliferative disorders are frequently involved in renal complications, including a specific proximal tubule impairment, Fanconi's syndrome. The latter is characterized in most cases by intracellular crystallization including a light-chain variable-domain fragment which resists lysosomal proteases. Bence-Jones protein (BJP) DEL was isolated from a patient with myeloma-associated Fanconi's syndrome. The crystal structure of this human kappa immunoglobulin light-chain noncovalent dimer was determined using molecular replacement with the structure of molecule REI, as the variable domain, and that of BJP LOC as the constant domain. To our knowledge, DEL is the first complete kappa BJP structure described to date. The R-factor is 20.7% at 2.8 A resolution. The BJP DEL dimer was compared with other light-chain dimers and with Fab fragments with a kappa light chain. Although the domain-folding pattern was similar, the relative positions of the constant domains differed. BJP DEL showed a noncanonical quaternary structural arrangement which may be attributable to the poor CL-CL affinity and lack of an interchain disulfide bridge, combined with the conformational editing effect of the crystal-packing forces. Our results suggest that, in the absence of a disulfide bridge, most BJP CLs are probably mobile in solution. This may explain their high susceptibility to proteases and the absence of naturally occurring crystals for these dimers. Furthermore, these findings of an unusual quaternary structure of an immunoglobulin light-chain association extend our knowledge about the large and highly diverse structures of the immunoglobulin superfamily.
Subject(s)
Bence Jones Protein/chemistry , Immunoglobulin Constant Regions/immunology , Immunoglobulin Light Chains/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Dimerization , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Sequence Alignment , Water/metabolismABSTRACT
The objective of this study was to further characterize the clinical and immunopathologic features of heavy chain deposition disease (HCDD), a recently described entity. Four patients were diagnosed as having HCDD on a kidney biopsy. All presented with nodular glomerulosclerosis with deposition of gamma1 heavy chains lacking CH1 epitopes, but without light chains. Two different patterns were observed in the serum. First, patients 1 and 2 had a circulating monoclonal IgGlambda containing a short gamma1 heavy chain lacking CH1 epitopes, with an apparent molecular weight of 40 kD consistent with a complete CH1 deletion. Biosynthetic experiments also showed that the deleted heavy chain was produced in excess compared with light chains, and was secreted in vitro together with half Ig molecules, although these abnormal components were not detected by Western blot analysis of whole serum. Second, patients 3 and 4 had a circulating monoclonal IgG1lambda with an apparently normal, nondeleted heavy chain subunit, but serum fractionation followed by immunoblotting revealed an isolated monoclonal gamma1 chain lacking CH1 epitopes. These data strongly suggest that renal deposition of a CH1-deleted heavy chain circulating in low amounts in the serum as a free unassembled subunit is a major feature of HCDD. The CH1 deletion is most likely responsible for the premature secretion in blood of the heavy chain by a clone of plasma cells.
Subject(s)
Bone Marrow/pathology , Glomerulosclerosis, Focal Segmental/pathology , Heavy Chain Disease/pathology , Immunoglobulin A/analysis , Immunoglobulin G/analysis , Adult , Aged , Basement Membrane/ultrastructure , Biopsy, Needle , Female , Glomerular Mesangium/ultrastructure , Glomerulosclerosis, Focal Segmental/blood , Glomerulosclerosis, Focal Segmental/immunology , Glomerulosclerosis, Focal Segmental/urine , Heavy Chain Disease/complications , Humans , Immunoblotting , Immunohistochemistry , Immunoproliferative Small Intestinal Disease/complications , Immunoproliferative Small Intestinal Disease/pathology , Male , Microscopy, Electron , Middle Aged , Sensitivity and SpecificityABSTRACT
Light chain deposition disease is a severe complication of certain immunoproliferative disorders, due to the secretion of a monoclonal light chain which precipitates close to basement membranes of several tissues. A kappa isotype restriction and an unusual frequency of a variable region subgroup (VkappaIV) suggest that precise structural features govern the propensity of pathogenic light chains to precipitate in extracellular spaces. We studied primary structures of light chains from six patients with light chain deposition disease in comparison with light chains from other pathological conditions. Sequence alignment revealed the presence of certain amino acids only in light chain deposition disease, in particular non-polar replacing hydrophilic residues. To determine the role of these residues, structures of the variable domain from four kappa chains belonging to VkappaI and VkappaIV subgroups responsible for deposition disease were modeled using known immunoglobulins as templates. The most evident structural features shared by all pathogenic light chains were hydrophobic residues exposed to the solvent in complementarity determining regions 1 or 3. In contrast to immunoglobulin light chain-related amyloidosis, where deposition of organized material might be due to electrostatic interactions between light chain dimers, hydrophobic interactions could enhance amorphous precipitation in non-amyloid light chain deposition disease.
Subject(s)
Hypergammaglobulinemia/metabolism , Immunoglobulin Light Chains/chemistry , Amino Acid Sequence , Databases, Factual , Models, Molecular , Molecular Sequence Data , Sequence Alignment , Stereoisomerism , Surface PropertiesABSTRACT
Certain monoclonal Ig light chains (LC) are responsible for marked disturbances of proximal tubule cell functions (Fanconi's syndrome, FS). In patients with FS, intracellular crystal-like inclusions containing LC determinants are commonly found in plasma cells, macrophages, and renal tubular cells. In an attempt at understanding the pathogenesis of myeloma-associated FS, we recently determined the first complete primary sequence of a kappa-LC (CHEB) responsible for the disease. We now report on the primary structure of three other kappa-LC of the V kappa l variability subgroup associated with FS (TRE, TRO, and DEL). After PCR amplification, cDNA encoding these LC were sequenced. CHEB, TRE, and TRO LC genes were found to be highly homologous to the same germline gene O2/O12. These patients had numerous intracellular crystals, whereas the fourth patient, DEL, had no detectable crystals. The LC from the latter patient was homologous to another germline gene, O8/O18. Comparison of these LC sequences to previously reported LC of the V kappa l subgroup allowed identification of a number of unusual amino acid substitutions in the V region that had rarely or never been previously described at the corresponding positions. Some of these unusual substitutions affect highly conserved amino acids located either in an external loop (residue 30) or in inner (residues 48 and 55) and outer (positions 63 and 72) beta-sheets that may be important for the structure and binding properties of the kappa-chains. These and several other substitutions, some of them shared with amyloidogenic kappa-LC, could induce conformational alterations and represent a determinant pathogenic factor.
Subject(s)
Fanconi Syndrome/immunology , Immunoglobulin Light Chains/immunology , Immunoglobulin Variable Region/immunology , Amino Acid Sequence , Base Sequence , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Fanconi Syndrome/pathology , Humans , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Kidney/immunology , Kidney/pathology , Kidney/ultrastructure , Microscopy, Electron , Molecular Sequence Data , Sequence AlignmentABSTRACT
Although various programs are available to extract all the information included in protein sequence databases, none is dedicated to immunoglobulins. For this purpose, we designed a program, SUBIM, which is adapted to the Kabat database specialized in immunoglobulin sequences. Besides all the possibilities of any database searching program, SUBIM analyses new sequences of variable regions and determines the variability subgroup they belong to. It also numbers the new sequence according to the system established by Kabat and co-workers for an easier comparison with the other immunoglobulins, thus realizing an automatic alignment with other members of a given type of immunoglobulin chain. This program is largely machine independent and requires very little memory, and should help biochemists concerned with new immunoglobulin sequences.
Subject(s)
Databases, Factual , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Software , Amino Acid Sequence , Animals , Consensus Sequence , Evaluation Studies as Topic , Humans , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The variability subgroup of human monoclonal kappa chains purified from urine in 3 consecutive patients with myeloma associated light chain deposition disease was determined from amino acid sequences of their first framework regions (FR1). N-glycosylation was searched for by N-glycosidase F treatment. These data together with our previously published results, indicate the pathogenic potential of the rare V kappa IV subgroup and confirm the absence of detectable serum and urine free monoclonal light chains when they are N-glycosylated.