5alpha-reductase activity of genital and nongenital skin fibroblasts from patients with 5alpha-reductase deficiency, androgen insensitivity, or unknown forms of male pseudohermaphroditism

Serially subcultured fibroblast strains from genital (foreskin, labium majus) skin, as a group, have considerably more steroid (testosterone) 5alpha-reductase activity than those form nongenital skin. Foreskin strains varied 40-fold and labial strains even more. Labial strains overlapped nongenital strains of either sex more frequently than did prepuce strains. The activity of foreskin strains from two siblings with proven 5alpha-reductase deficiency was clearly lower than that of any of 18 control stains. The comparative behavior of the various strain types indicates that labial and nongenital strains should not be used to support a clinical suspicion of male pseudohermaphroditism due to 5alpha-reductase deficiency. The activities of labial strains from patients with complete androgen insensitivity (testicular feminization) - five with the receptor-negative variety and two with the receptor-positive type - were as variable as those of control labial strains. The decreased 5alpha-reductase activity observed in fresh skin slices of some patients is probably and expression of their functional estrogen/androgen imbalance in vivo.     

Phenotypic classification of male pseudohermaphroditism due to steroid 5α-reductase 2 deficiency

Conversion of testosterone (T) to dihydrotestosterone (DHT) in genital tissue is catalysed by the enzyme 5α-reductase 2, which is encoded by the SRD5A2 gene. The potent androgen DHT is required for full masculinization of the external genitalia. Mutations of the SRD5A2 gene inhibit enzyme activity, diminish DHT formation, and hence cause masculinization defects of varying degree. The classical syndrome, formerly described as pseudovaginal perineoscrotal hypospadias, is characterized by a predominantly female phenotype at birth and significant virtilization without gynecomastia at puberty. We investigated nine patients with steroid 5α-reductase 2 deficiency (SRD). Phenotypes, which were classified according to the severity of the masculinization defect, varied between completely female (SRD type 5), predominantly female (SRD type 4), ambiguous (SRD type 3), predominantly male with micropenis and hypospadias (SRD type 2), and completely male without overt signs of undermasculinization (SRD type 1). T/DHT-ratios were highly increased (>50) in the classical syndrome (SRD type 5), but variable in the less severe affected patients (SRD types 1–4) (14–35). Mutations in the SRD5A2 gene had been characterized using PCR-SSCP analysis and direct DNA sequencing. A small deletion was encountered in two patients, while all other patients had single base mutations which result in amino acid substitutions. We conclude that phenotypes may vary widely in patients with SRD5A2 gene mutations spanning the whole range from completely female to normal male without distinctive clinical signs of the disease. Hence, steroid 5α-reductase deficiency should be considered not only in sex reversed patients with female or ambiguous phenotypes, but also in those with mild symptoms of undermasculinization as encountered in patients with hypospadias and/or micropenis. A classification based on the severity of the masculinization defect may be used for correlation of phenotypes with enzyme activities and genotypes, and for comparisons of phenotypes between different patients as the basis for clinical decisions to be made in patients with pseudohermaphroditism due to steroid 5α-reductase 2 deficiency. © 1996 Wiley-Liss, Inc.     

Molecular analysis of the 5α-steroid reductase type 2 gene in a family with deficiency of the enzyme

This report describes the identification of a point mutation in the 5α-reductase type 2 (5α-SR2) gene from a family in which both sibs (6 and 3 years old) have steroid 5α-reductase 2 deficiency. The five exons of the gene were individually amplified by the polymerase chain reaction (PCR) and analysed for single-strand conformation polymorphisms (SSCP) to detect mutations. Direct sequencing of the mutant PCR products demonstrated a single C→T mutation, within exon 4, changing codon 227 from CGA (Arg) to TGA (premature termination signal). Both patients were homozygous for the mutation, but their parents were heterozygous. These results suggest that the mutation at codon 227 impairs normal 5α-SR2 function, thus leading to the phenotypical expression of this rare enzymatic defect. Am. J. Med. Genet. 69:69–72, 1997. © 1997 Wiley-Liss, Inc.     

New frameshift mutation in the 5α-reductase type 2 gene in a Brazilian patient with 5α-reductase deficiency

Male pseudohermaphroditism caused by steroid 5α-reductase deficiency is an autosomal recessive disorder. The enzyme steroid 5α-reductase 2 (encoded by the SRD5A2 gene) catalyses the conversion of testosterone to dihydrotestosterone, which is required for normal differentiation of the external male genitalia. This report describes the molecular analysis of the 5α-reductase type 2 gene in a Brazilian patient who was raised as a female, underwent a reversal of gender role behavior, and is now a married man. This patient is a compound heterozygote bearing an A→G mutation within exon 2, changing codon 126 from Glu to Arg on one allele and a novel single base deletion (418delT) causing a frameshift mutation at codon 140 in the same exon, on the other allele. This last mutation probably leads to the synthesis of a truncated protein, because a premature termination signal is created at codon 159. Am. J. Med. Genet. 87:221–225, 1999. © 1999 Wiley-Liss, Inc.     

Homozygous mutation (A228T) in the 5α-reductase type 2 gene in a boy with 5α-reductase deficiency: Genotype–phenotype correlations

The molecular basis of a patient with 5α-reductase deficiency was investigated in this study. This disease is a rare form of male pseudohermaphroditism with virilization during puberty. The child was raised as a girl, but had a male gender identity early in life. The diagnosis was set at the age of 13 years when the virilization process began. Hypospadias repair was performed and he changed to a male gender. DNA sequence analysis disclosed a homozygous mutation in exon 4 of the 5α-reductase type 2 gene, alanine 228 for threonine. The heterozygous parents are first cousins of Pakistani origin. Am. J. Med. Genet. 80:269–272, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of R5‐tropic HIV type‐1 replication in CD4+ natural killer T cells by γδ T lymphocytes

After the development of highly active anti‐retroviral therapy, it became clear that the majority of emergent HIV‐1 is macrophage‐tropic and infects CD4⁺, CCR5‐expressing cells (R5‐tropic). There are three distinct cell populations, R5‐tropic, HIV‐1‐susceptible CD4⁺ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue‐associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4⁺ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4⁺ T cells derived from circulating PBMCs, and that R5‐tropic HIV‐1 expands efficiently in the CD4⁺ NKT cells. Moreover, when PBMCs depleted of CD8α⁺ cells were stimulated in the presence of α‐galactosylceramide (α‐GalCer) and R5‐tropic HIV‐1 [NL(AD8)], the production of HIV‐1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8β⁺ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8αα⁺ but not CD8αβ⁺ cells may have the ability to inhibit R5‐tropic HIV‐1 replication in CD4⁺ NKT cells. Here, we show that co‐culturing R5‐tropic HIV‐1‐infected CD4⁺ NKT cells with CD8αα⁺ γδ T cells, in particular Vγ1Vδ1 cells, but not with CD8αα⁺ NKT cells or CD8αα⁺ dendritic cells, inhibits HIV‐1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1α and 1β and RANTES. Collectively, these results indicate the importance of CD8αα⁺ γδ T cells in the control of R5‐tropic HIV‐1 replication and persistence in CD4⁺ NKT cells.     

Expression of HNF-1α and HNF-1β in various histological differentiations of hepatocellular carcinoma

Hepatic nuclear factor 1 (HNF-1) regulates genes in a hepatocyte-specific manner. It has been previously reported that the ratio of HNF-1α and HNF-1β mRNA is related to histological differentiation hepatocellular carcinoma (HCC). In this study, the expression levels of the HNF-1α and HNF-1β proteins were analysed relatively and quantitatively in various histologically differentiated HCC and surrounding non-cancerous tissues, and HNF-1α binding activity for the AT element of the B domain of the human α-fetoprotein enhancer was examined. Western blot analysis demonstrated that HNF-1α protein was expressed at a higher level in well-differentiated HCC tissues than in the surrounding non-HCC tissues; on the other hand, the HNF-1α protein was expressed at lower levels in moderately and poorly differentiated HCCs than in the surrounding non-HCC tissues. The levels of HNF-1β expression in well-differentiated and poorly differentiated HCCs were similar to and higher than those found in the respective surrounding non-cancerous portions. In binding assays, HNF-1 binding activity was high in well-differentiated HCC and lower in moderately and poorly differentiated HCCs. Most well-differentiated HCC cases showed immunohistochemical expression of HNF-1α. These findings show that poor histological differentiation of HCC correlates with decreases in the level and activity of HNF-1α proteins. © 1998 John Wiley & Sons, Ltd.     

Deletion of the α2A/α2C‐adrenoceptors accelerates cutaneous wound healing in mice

The α2‐adrenoceptors regulate the sympathetic nervous system, controlling presynaptic catecholamine release. However, the role of the α2‐adrenoceptors in cutaneous wound healing is poorly understood. Mice lacking both the α2A/α2C‐adrenoceptors were used to evaluate the participation of the α2‐adrenoceptor during cutaneous wound healing. A full‐thickness excisional lesion was performed on the dorsal skin of the α2A/α2C‐adrenoceptor knockout and wild‐type mice. Seven or fourteen days later, the animals were euthanized and the lesions were formalin‐fixed and paraffin‐embedded or frozen. Murine skin fibroblasts were also isolated from α2A/α2C‐adrenoceptor knockout and wild‐type mice, and fibroblast activity was evaluated. The in vivo study demonstrated that α2A/α2C‐adrenoceptor depletion accelerated wound contraction and re‐epithelialization. A reduction in the number of neutrophils and macrophages was observed in the α2A/α2C‐adrenoceptor knockout mice compared with wild‐type mice. In addition, α2A/α2C‐adrenoceptor depletion enhanced the levels of nitrite and hydroxyproline, and the protein expression of transforming growth factor‐β and vascular endothelial growth factor. Furthermore, α2A/α2C‐adrenoceptor depletion accelerated blood vessel formation and myofibroblast differentiation. The in vitro study demonstrated that skin fibroblasts isolated from α2A/α2C‐adrenoceptor knockout mice exhibited enhanced cell migration, α‐smooth muscle actin _protein expression and collagen deposition compared with wild‐type skin fibroblasts. In conclusion, α2A/α2C‐adrenoceptor deletion accelerates cutaneous wound healing in mice.     

The stimulatory effect of α‐melanotropin on progesterone release from rat granulosa cells is inhibited by interleukin‐1β and by tumour necrosis factor‐α

Aim: Several studies have shown that a variety of peptides and cytokines are involved in ovarian regulatory mechanisms; however, their exact function is still unclear. In this work we study whether the administration of peptide α‐melanotropin and the cytokines interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) on their own modify the release of progesterone in cultured granulosa cells (GC) from pro‐oestrous rats. We also investigate an interaction between these cytokines and α‐melanotropin in the modulation of progesterone secretion. Methods: Granulosa cells were collected from the ovaries of female Wistar rats and cultured for up to 24 h in the presence of different concentrations of α‐melanotropin, cytokines or a combination of both. Progesterone concentration was measured by radioimmunoassay. Results: The addition of α‐melanotropin in a dose of 0.01 and 0.1 mm had no effect on progesterone release, whereas a dose of 1 mm significantly increased progesterone release (P < 0.01) compared with the control culture. Progesterone release was not modified when different concentrations of interleukin‐1β or TNF‐α were added to the cell cultures. However, when interleukin‐1β or TNF‐α were added simultaneously with 1 μm α‐melanotropin, a significant reduction (P < 0.01 for interleukin‐1β and P < 0.05 for TNF‐α) of the steroid release was found with respect to the α‐melanotropin‐treated group. Conclusions: These results lead us to suggest that, although α‐melanotropin stimulates progesterone release in pre‐ovulatory GC, this effect is blocked by the presence of interleukin‐1β or TNF‐α.     

AMPKα2 reduces renal epithelial transdifferentiation and inflammation after injury through interaction with CK2β

TGFβ1/Smad, Wnt/β‐catenin and snail1 are preferentially activated in renal tubular epithelia after injury, leading to epithelial–mesenchymal transition (EMT). The stress response is coupled to EMT and kidney injury; however, the underlying mechanism of the stress response in EMT remains elusive. AMP‐activated protein kinase (AMPK) signalling is responsive to stress and regulates cell energy balance and differentiation. We found that knockdown of AMPKα, especially AMPKα2, enhanced EMT by up‐regulating β‐catenin and Smad3 in vitro. AMPKα2 deficiency enhanced EMT and fibrosis in a murine unilateral ureteral obstruction (UUO) model. AMPKα2 deficiency also increased the expression of chemokines KC and MCP‐1, along with enhanced infiltration of inflammatory cells into the kidney after UUO. CK2β interacted physically with AMPKα and enhanced AMPKα Thr172 phosphorylation and its catalytic activity. Thus, activated AMPKα signalling suppresses EMT and secretion of chemokines in renal tubular epithelia through interaction with CK2β to attenuate renal injury. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Expression of androgen-responsive properties in human skin fibroblast strains of genital and nongenital origin

pecific 5-dihydrotestosterone (DHT) binding capacity (B_max) has been determined for human skin fibroblast strains from nongenital areas of males and females (N=8), as well as prepuce and labium majus (N=9). Genital strains had a mean three times that of nongenital ones (32 vs. 11 fmollmg cell protein). There were no sex differences. Variation among strains was not simply correlated with donor age; that within strains was unrelated to in vitro age. The lowest values for genital strains overlapped the nongenital ones; those of the nongenital strains approached the limit of detectability. These results parallel those for ⁴ -3-ketosteroid 5-reductase activity. Thus, serially cultured genital and nongenital skin fibroblasts express their relative differentiative ancestry as androgen target cells. This expression may affect the diagnosis of androgen insensitivity and certain inborn errors of metabolism; its variability is discussed in terms of clonal heterogeneity.     

α2,3-Sialyl and α1,3-fucosyltransferase-dependent synthesis of sialyl Lewis x,an essential oligosaccharide present on L-selectin counterreceptors,in cultured endothelial cells

Sialyl Lewis x (sLex) oligosaccharides have been shown to be present in counterreceptors for L-selectin. We and others have previously shown that high endothelial cells in lymph nodes and at sites of inflammation express sLex. Here we show that also cultured human umbilical vein endothelial cells (HUVEC) express sLex on their cell surface. This oligosaccharide is formed by sequential action of α2,3-sialyl- (α2,3-ST) and α1,3-fucosyltransferases (α1,3-FT) on N-acetyllactosamine. At least two of the several α2,3-ST and four of the several α1,3-FT are present in HUVEC. In functional assays both α2,3-ST and α1,3-FT activities were observed in HUVEC lysates with exogenous lactosamine and sialyllactosamine acceptors, leading to the generation of the sialyllactosamine and sLex sequences, respectively. TNF stimulation increased the level of mRNA expression of FT VI, and the α1,3-FT activity in HUVEC. Taken together these data show that endothelial cells express sLex and that they possess mRNA as well as enzyme activities of several α2,3-ST and α1,3-FT necessary in the final steps of sLex synthesis. Furthermore, inflammatory cytokines such as TNF can enhance transferase activities relevant in generating putative L-selectin counterreceptors.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Epitopes recognized by neutralizing therapy-induced human anti-interferon-α antibodies are localized within the N-terminal functional domain of recombinant interferon-α2

During prolonged recombinant interferon (rIFN)-α2 therapy, a minority of patients develop high-titer neutralizing IFN-α antibodies. Sera from nine IFN-α antibody-positive patients were studied to characterize the specificity of anti-IFN-α neutralizing antibodies by their ability to inhibit the antiviral and antiproliferative activity of different rIFN-α subtypes and rIFN-α1/α2 hybrids. These therapy-induced antibodies (Tab) were compared with IFN-α-specific autoantibodies (Aab) from two patients with systemic lupus erythematosus who had never received any exogenous IFN-α. Although IFN-α subtypes are closely related in structure, Tab inhibited the antiviral activity of only recombinant (r)IFN-α2 and rIFN-α6, but not or slightly that of rIFN-α1, -α7, -α8 and -α14. Furthermore, of four different rIFN-α1/α2 hybrids tested, Tab inhibited only those which contained the N-terminal residues 17–64 of rIFN-α2. Comparison of the primary sequences of neutralized and not neutralized subtypes suggests an epitope involving the residues 22–31 of IFN-α2 is recognized. Thus, Tab block rIFN-α2 by reacting with only one of two functional domains. In contrast, Aab possessed a broad specificity and neutralized both the antiviral and antiproliferative activity of rIFN-α2, -α6, -α7, -α8 and -α14. They also neutralized all four rIFN-α1/α2 hybrids tested. These data demonstrate that Tab are highly specific for the therapeutic IFN-α subtype and specifically neutralize rIFN-α2 by binding to its N-terminal functional domain.     

Interleukin-1β (IL-1β) Is Increased in the Follicular Fluids of Patients With Premature Luteinization

PROBLEM : Most, but not all, studies indicate that premature luteinization correlates with poor pregnancy outcome in in-vitro fertilization (IVF) programs. It remains unclear whether cytokines (IL-1β, TNFα), the established immune mediators, play a role in regulation or initiation of an abnormal follicular or embryo development in patients with premature luteinization. METHODS : Levels of cytokines (IL-1β, TNFα), estradiol (E2), progesterone (P4), and androstenedione (Aione) were examined in 18 preovulatory follicular fluid (FF) samples from patients with premature luteinization (group 1) and 37 FF samples from patients without premature luteinization (group 2). The number of oocytes recovered, fertilization rate, and pregnancy outcome were evaluated in these two groups. RESULTS : IL-1β (25.4±11.9 pg/ml, mean ±SD) and TNFα (13.4±10.7 pg/ml) were present in these FF samples. The mean level of IL-1β in group 1 was significantly higher than that in group 2 (37.3±12.3 vs. 20.0±7.6 pg/ml; P<0.00001) and the mean level of E2 was significantly lower in group 1 than that in group 2 (1064±686 vs. 1570±641 ng/ml; P=0.02). The levels of TNFα, P4, and A'ione showed no distinction between these two groups. There was no correlation between the levels of either IL-1β or TNFα and P4, E2 or A'ione. The fertilization rate in group 1 (62/77; 80%) was similar to that in group 2 (124/160;78%). Five of 7 patients in group 1 and seven of 20 patients in group 2 achieved pregnancy following embryo transfer. One of five pregnancies in group 1 aborted. CONCLUSION : The exaggerated levels of IL-1β in patients with premature luteinization may arise from accumulation of this cytokine owing to sustained high LH stimulation, and this may be a protective response to the abnormal LH surge and function to inhibit prematurely increased secretion of P4. These data indicate the important role of LH in the induction of IL-1β secretion and the possible regulatory action of IL-1β in luteinization. According to the diminution of E2 in group 1, there may be a subtle atretic process progressing in follicles primed with prematurely elevated LH. However, the detrimental effect of premature luteinization, if it exists, may work at the stage during or after implantation.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

Serum thymosin α 1 levels in patients with chronic inflammatory autoimmune diseases

Thymosin alpha 1 (Tα1) is a powerful modulator of immunity and inflammation. Despite years of studies, there are a few reports evaluating serum Tα1 in health and disease. We studied a cohort of healthy individuals in comparison with patients affected by chronic inflammatory autoimmune diseases. Sera from 120 blood donors (healthy controls, HC), 120 patients with psoriatic arthritis (PsA), 40 with rheumatoid arthritis (RA) and 40 with systemic lupus erythematosus (SLE), attending the Transfusion Medicine or the Rheumatology Clinic at the Policlinico Tor Vergata, Rome, Italy, were tested for Tα1 content by means of a commercial enzyme‐linked immunosorbent assay (ELISA) kit. Data were analysed in relation to demographic and clinical characteristics of patients and controls. A gender difference was found in the HC group, where females had lower serum Tα1 levels than males (P < 0·0001). Patients had lower serum Tα1 levels than HC (P < 0·0001), the lowest were observed in PsA group (P < 0·0001 versus all the other groups). Among all patients, those who at the time of blood collection were taking disease‐modifying anti‐rheumatic drugs (DMARD) plus steroids had significantly higher Tα1 levels than those taking DMARD alone (P = 0·044) or no treatment (P < 0·0001), but not of those taking steroids alone (P = 0·280). However, whichever type of treatment was taken by the patients, serum Tα1 was still significantly lower than in HC and there was no treatment‐related difference in PsA group. Further prospective studies are necessary to confirm and deepen these observations. They might improve our understanding on the regulatory role of Tα1 in health and disease and increase our knowledge of the pathogenesis of chronic inflammatory autoimmune diseases.