Steroidogenic enzyme activity in the rat testis following Leydig cell destruction by ethylene-1,2-dimethanesulphonate and during subsequent Leydig cell regeneration.

Ethylene-1,2-dimethanesulphonate (EDS) rapidly destroys Leydig cells in the rat testis, although repopulation occurs within 5-7 weeks. In this study we have examined the activity of testicular steroidogenic enzymes after Leydig cell destruction and during regeneration. This was designed to measure the contribution of cells, other than Leydig cells, to steroidogenic activity in the testis, and to determine whether changes in steroidogenic enzyme activity during Leydig cell regeneration after EDS parallel those which occur during normal Leydig cell development. The enzymes studied are those responsible for androgen synthesis and metabolism in the testis. Adult male Wistar rats (300-350 g) were injected with EDS (100 mg/kg, i.p.) and testicular steroidogenic enzyme activity was measured on days 0, 3, 7, 14, 21 and 35. On day 3, when no Leydig cells remain in the testis, cholesterol side-chain cleavage (CSCC) activity, per testis, declined to undetectable levels, while 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17 alpha-hydroxylase retained only 0.04 and 0.15% of control activity respectively. In contrast, 17-ketosteroid reductase (17-KSR) and 5 alpha-reductase retained 33 and 10% of control activity respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leydig cell apoptosis after the administration of ethane dimethanesulfonate to the adult male rat is a Fas-mediated process.

Leydig cells undergo apoptosis in response to the cytotoxin ethane dimethanesulfonate (EDS), with numbers declining at 12-18 h and maximal apoptosis at 24 h postinjection. The Bcl-2 family members, Bcl-2, Bcl-xl, and Bax, appear not to be involved in this process. To further investigate this phenomena, a single dose of EDS was administered to adult rats to induce the killing of Leydig cells. The interstitial cells were examined up to 3 days after EDS administration by Western blot analysis for the Bcl-2 family members (Bak and Bcl-w). Western blotting showed that Bak expression in the interstitial cell preparations was unchanged after EDS, and immunohistochemistry showed that it was not up-regulated in Leydig cells in response to EDS. Bcl-w expression in the Leydig cells and interstitial cell preparations was unchanged until 48 h when it became undetectable, suggesting that Leydig cell-associated Bcl-w is not involved in initiating apoptosis. We also investigated the role of the Fas system in Leydig cell apoptosis. Both Fas receptor and Fas ligand protein levels increased after EDS, peaking at 12-18 h and declining thereafter. Fas receptor and ligand were shown by immunohistochemistry to be present in Leydig cells, and after EDS all Leydig cells became strongly positive for both proteins. The intensity of staining increased in the early stages of apoptosis and decreased as the nuclear morphology became more fragmented. These data suggest that Bcl-2 family members are not involved in Leydig cell apoptosis after EDS administration. However, up-regulation of the Fas system does occur, implicating activation of Fas receptor in the induction of Leydig cell apoptosis.     

Immunologic regulation of E. coli K1 by serum from neonatal rats is enhanced following intraperitoneal administration of human IgG

The effect of 1500 mg/kg intraperitoneal human IgG on the capacity of neonatal rat serum to opsonize and to kill Escherichia coli K1 and to deposit IgG and C3 onto this organism was investigated. Unlike serum from neonatal rats injected with albumin, serum from neonatal rats injected with human IgG opsonized and killed E. coli K1 efficiently. Heat treatment abolished the bactericidal effect of serum from IgG recipients, suggesting a role for complement. A radioimmunobinding assay demonstrated that the capacity of neonatal rat serum to deposit IgG and C3 onto E. coli K1 was impaired. However, intraperitoneal human IgG enabled serum from neonatal rats to deposit human IgG onto the bacteria and enhanced C3 deposition to a level equivalent to that observed with adult rat serum. Therefore, neonatal rats have a functionally competent classic pathway of complement. Human IgG ameliorates the opsonic and bacteriolytic inadequacies of neonatal rat serum that result primarily from a deficiency of antibodies to E. coli.     

Influence of long-term dietary administration of procymidone, a fungicide with anti-androgenic effects, or the phytoestrogen genistein to rats on the pituitary-gonadal axis and Leydig cell steroidogenesis

Procymidone is a fungicide with anti-androgenic properties, widely used to protect fruits from fungal infection. Thereby it contaminates fruit products prepared for human consumption. Genistein-containing soy products are increasingly used as food additives with health-promoting properties. Therefore we examined the effects of long-term dietary administration (3 months) of the anti-androgen procymidone (26.4 mg/animal per day) or the phytoestrogen genistein (21.1 mg/animal per day) to rats on the pituitary-gonadal axis in vivo, as well as on Leydig cell steroidogenesis and on spermatogenesis ex vivo. The procymidone-containing diet elevated serum levels of LH and testosterone and, furthermore, Leydig cells isolated from procymidone-treated animals displayed an enhanced capacity for producing testosterone in response to stimulation by hCG or dibutyryl cAMP, as well as elevated expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450 scc) and cytochrome P450 17alpha (P450c17). In contrast, the rate of DNA synthesis during stages VIII and IX of spermatogenesis in segments of seminiferous tubules isolated from genistein-treated rats was decreased without accompanying changes in the serum level of either LH or testosterone. Nonetheless, genistein did suppress the ex vivo steroidogenic response of Leydig cells to hCG or dibutyryl cAMP by down-regulating their expression of P450 scc. Considered together, our present findings demonstrate that long-term dietary administration of procymidone or genistein to rats exerts different effects on the pituitary-gonadal axis in vivo and on Leydig cell steroidogenesis ex vivo. Possibly as a result of disruption of hormonal feedback control due to its anti-androgenic action, procymidone activates this endocrine axis, thereby causing hyper-gonadotropic activation of testicular steroidogenesis. In contrast, genistein influences spermatogenesis and significantly inhibits Leydig cell steroidogenesis ex vivo without altering the serum level of either LH or testosterone.     

Assessment of acute spinal inflammation in patients with ankylosing spondylitis by magnetic resonance imaging: a comparison between contrast enhanced T1 and short tau inversion recovery (STIR) sequences

OBJECTIVES: To compare the performance of two different MRI sequences-T(1) weighted, fat saturated, spin echo after application of contrast medium, and short tau inversion recovery (STIR) sequences-to detect spinal inflammation in patients with ankylosing spondylitis (AS). METHODS: Both MRI sequences were performed in 38 patients with active AS and compared using the MRI activity scoring system, ASspiMRI-a. One vertebral unit (VU) was defined as the region between two virtual lines drawn through the middle of each vertebral body. RESULTS: Intraclass correlation coefficients were excellent-0.91 and 0.86 for the Gd-DTPA and STIR sequences, respectively. The overall correlation of the single MRI scores for both sequences was also good (r = 0.84, p = 0.01). The intrarater variance was 6.71 and 9.41 and the interrater variance was 13.16 and 19.04 for the Gd-DTPA and STIR sequences, respectively. The smallest detectable distance was 4.7 and 5.6 for the Gd-DTPA and STIR sequences, respectively. The concordance rate for both sequences was 83.5% (range 80.5-87.7% in the three spinal segments). Inflammatory spinal lesions were found in 10.1% of the VUs in the STIR sequence but not in the T(1)/Gd-DTPA sequence, while the T(1)/Gd-DTPA sequence showed inflammatory lesions in 6.4% of the VUs that were found normal by STIR. CONCLUSIONS: Both MRI techniques can evaluate active spinal lesions in patients with AS. More spinal lesions are detected by the STIR sequence, but the reliability between readings and readers is better for the Gd-DTPA sequence. The ASspiMRI-a is a reliable instrument for evaluating acute spinal changes in AS.     

NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc epsilon RI aggregation

Aggregation of high-affinity receptors for immunoglobulin E (Fc epsilon RI) on the surface of mast cells results in degranulation, a response that is potentiated by binding of stem cell factor (SCF) to its receptor Kit. We observed that one of the major initial signaling events associated with Fc epsilon RI-mediated activation of human mast cells (HuMCs) is the rapid tyrosine phosphorylation of a protein of 25 to 30 kDa. The phosphorylation of this protein was also observed in response to SCF. This protein was identified as non-T-cell activation linker (NTAL), an adaptor molecule similar to linker for activated T cells (LAT). Unlike the Fc epsilon RI response, SCF induced NTAL phosphorylation in the absence of detectable LAT phosphorylation. When SCF and antigen were added concurrently, there was a marked synergistic effect on NTAL phosphorylation, however, SCF did not enhance the phosphorylation of LAT induced by Fc epsilon RI aggregation. Fc epsilon RI- and SCF-mediated NTAL phosphorylation appear to be differentially regulated by Src kinases and/or Kit kinase, respectively. Diminution of NTAL expression by silencing RNA oligonucleotides in HuMCs resulted in a reduction of both Kit- and Fc epsilon RI-mediated degranulation. NTAL, thus, appears to be an important link between the signaling pathways that are initiated by these receptors, culminating in mast cell degranulation.     

Development of cytotoxic islet cell antibodies in rats following damage of the pancreas by complete Freund's adjuvant combined with a nondiabetogenic dose of streptozotocin

The possible relationship between destruction of pancreatic beta cells and islet cell surface antibodies (ICSA) was examined in a rat model using complete Freund's adjuvant (CFA), a lymphocyte activator, in combination with the beta cell toxin, streptozotocin (STZ). In addition to this treatment, the rat insulinoma cell line, RIN5AH, as a readily accessible source of insulin-producing cells, was utilized to potentiate the production of ICSA. Intraperitoneal injections of CFA to male Lewis rats, followed 24 h later by a single nondiabetogenic dose of STZ, produced a 47% (p less than 0.01) reduction in pancreatic insulin content associated with degranulation and necrosis of insulin-immunoreactive cells. Eight weeks after treatment, ICSA were detectable that mediated the complement-dependent lysis of neonatal rat islet cells. Injections of RIN5AH cells, following treatment with CFA/STZ, did neither increase the severity of histopathological changes in the exocrine pancreas nor the extent of beta cell necrosis, but gave rise to higher levels of cytotoxic ICSA. Immunization with RIN cells alone, although increasing ICSA levels above those of the other experimental groups, produced no major histopathological changes. These results indicate that ICSA are the consequence of beta cell damage, and they are not capable of promoting or initiating beta cell necrosis in this model.     

Development of cytotoxic islet cell antibodies in rats following damage of the pancreas by complete freund’s adjuvant combined with a nondiabetogenic dose of streptozotocin

The possible relationship between destruction of pancreatic beta cells and islet cell surface antibodies (ICSA) was examined in a rat model using complete Freund’s adjuvant (CFA), a lymphocyte activator, in combination with the beta cell toxin, streptozotocin (STZ). In addition to this treatment, the rat insulinoma cell line, RIN5AH, as a readily accessible source of insulin-producing cells, was utilized to potentiate the production of ICSA. Intraperitoneal injections of CFA to male Lewis rats, followed 24 h later by a single nondiabetogenic dose of STZ, produced a 47% (p<0.01) reduction in pancreatic insulin content associated with degranulation and necrosis of insulin-immunoreactive cells. Eight weeks after treatment, ICSA were detectable that mediated the complement-dependent lysis of neonatal rat islet cells. Injections of RIN5AH cells, following treatment with CFA/ STZ, did neither increase the severity of histopathological changes in the exocrine pancreas nor the extent of beta cell necrosis, but gave rise to higher levels of cytotoxic ICSA. Immunization with RIN cells alone, although increasing ICSA levels above those of the other experimental groups, produced no major histopathological changes. These results indicate that ICSA are the consequence of beta cell damage, and they are not capable of promoting or initiating beta cell necrosis in this model.     

HISS-dependent insulin resistance (HDIR) in aged rats is associated with adiposity, progresses to syndrome X, and is attenuated by a unique antioxidant cocktail

The hypotheses were: HISS-dependent insulin resistance (HDIR) accounts for insulin resistance that occurs with aging; HDIR is the initiating metabolic defect that leads progressively to type 2 diabetes and the metabolic syndrome; a synergistic antioxidant cocktail in chow confers protection against HDIR, subsequent symptoms of diabetes, and the metabolic syndrome. Male Sprague Dawley rats were tested at 9, 26, and 52 weeks to determine their dynamic response to insulin, the HISS (hepatic insulin sensitizing substance)-dependent component of insulin action, and the HISS-independent (direct) insulin action using a dynamic insulin sensitivity test. In young rats, the HISS component accounted for 52.3+/-2.1% of the response to a bolus of insulin (50mU/kg) which decreased to 29.8+/-3.4% at 6 months and 17.0+/-2.7% at 12 months. HISS action correlated negatively with whole body adiposity and all regional fat depots (r(2) = 0.67-0.87). The antioxidants (vitamin C, vitamin E, and S-adenosylmethionine) conferred protection of HISS action, fat mass at all sites, blood pressure, postprandial insulin and glucose. Data are consistent with the hypotheses. Early detection and therapy directed towards treatment of HDIR offers a novel therapeutic target.     

Underactive bladder in aging rats is associated with a reduced number of serotonin‐expressing cells in the urethra and is improved by serotonin application to the urethra

The aim of this study was to determine whether aging‐related detrusor underactivity (DU) involves a decrease in 5‐hydroxytryptamine (5‐HT‐positive)‐expressing urethral cells and whether 5‐HT stimulation of urethral sensory fibers improves detrusor function. Cystometries were performed in young (6 months) and aged (18–24 months) female Wistar rats. Aged rats with voiding contractions (VC) that were 2SD below the mean of those in young rats were considered to have DU. Bladder voiding efficiency (BVE) was calculated during saline or 5‐HT solution cystometries. Rats were perfusion‐fixed with a fixative solution (paraformaldehyde, PFA 4%) through the circulatory system and the urethra sectioned to count the number of 5‐HT‐immunoreactive (IR) cells. Isovolumetric cystometry was performed while irrigating the urethra with saline or 5μM‐HT solution. Two‐tailed unpaired t tests were used to determine the significance of differences. In aged DU rats, the mean (±SD) VC frequency was 0.24 ± 0.07 per minute, with an amplitude of 15 ± 3 cm H₂O. The mean (±SD) number of 5‐HT‐IR cells in the urethra of aged DU and young rats was 90 ± 11 and 182 ± 25, respectively (P < 0.01). 5‐HT improved the mean (±SD) BVE of aged DU rats from 49 ± 3% to 78 ± 2% (P < 0.001). In isovolumetric cystometries, detrusor pressure during irrigation of the urethra with saline was 18 ± 1 cm H₂O, compared with 39 ± 2 cm H₂O during irrigation with 5‐HT solution (P < 0.05). In rats, DU associated with aging is accompanied by a decrease in the number of 5‐HT‐positive cells. The results suggest that decreased 5‐HT availability decreases urethral sensory fiber excitation, leading to a decrease the number of effective VC.     

Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.

We demonstrate here that coexpression of ROMK2, an inwardly rectifying ATP-sensitive renal K+ channel (IKATP) with cystic fibrosis transmembrane regulator (CFTR) significantly enhances the sensitivity of ROMK2 to the sulfonylurea compound glibenclamide. When expressed alone, ROMK2 is relatively insensitive to glibenclamide. The interaction between ROMK2, CFTR, and glibenclamide is modulated by altering the phosphorylation state of either ROMK2, CFTR, or an associated protein, as exogenous MgATP and the catalytic subunit of protein kinase A significantly attenuate the inhibitory effect of glibenclamide on ROMK2. Thus CFTR, which has been demonstrated to interact with both Na+ and Cl- channels in airway epithelium, modulates the function of renal ROMK2 K+ channels.     

Induction of CD11a/leukocyte function antigen-1 and CD54/intercellular adhesion molecule-1 on hairy cell leukemia cells is accompanied by enhanced susceptibility to T-cell but not lymphokine-activated killer-cell cytotoxicity.

Some B-cell neoplasms, including hairy cell leukemia (HCL), lack expression of the adhesion molecule leukocyte function antigen-1 (LFA-1/CD11a). Additionally, HCL cells express relatively low amounts of intercellular adhesion molecule-1 (ICAM-1/CD54) and may therefore be an inappropriate target for recognition by T cells or lymphokine-activated killer (LAK) cells. We tested whether these molecules were inducible on HCL cells and if induction would lead to enhanced susceptibility to lysis by LAK cells or cytolytic T cells. CD11a expression was induced by incubation with interferon-alpha (IFN-alpha) or interleukin-4. CD54 was induced by culturing the cells irrespectively of the addition of cytokines. Expression of CD11a and CD54 did not enhance susceptibility to either autologous or allogeneous LAK cells. However, induction of these adhesion molecules was accompanied by enhanced susceptibility to lysis by cytotoxic T lymphocyte clones. This lysis could be reversed by the addition of anti-CD11a and anti-CD54 antibodies. Finally, we monitored the expression of CD11a and CD54 on HCL cells from patients during IFN-alpha therapy. In one of four patients monitored, we observed rapid in vivo induction of CD11a and CD54 on the leukemic cells during IFN-alpha therapy. These studies provide a model for studying immunosurveillance in HCL.     

T cell response to the cytomegalovirus major capsid protein (UL86) is dominated by helper cells with a large polyfunctional component and diverse epitope recognition

T cells are crucial in controlling cytomegalovirus (CMV) infection. The CMV major capsid protein (UL86) is frequently recognized by these cells, but the nature of this response has not been explored in detail. In this study, healthy CMV-exposed individuals were examined, and ex vivo peptide stimulation of peripheral blood mononuclear cells and flow-cytometry were used to obtain data, including response prevalence, magnitude, functional profiles, and recognized epitopes. Of 24 subjects, 19 (79%) had a UL86-specific CD4 T cell response rate between 0.03% and 1.4%. This group of individuals exhibited a similar percentage of polyfunctional T cells in their UL86-specific and pp65-specific responses. A total of 8 CD4 T cell epitopes were identified. In contrast, CD8 T cell responses to UL86 were rare and small. UL86 is of interest for monitoring the response to CMV.     

Slow transitions between two conformational states of band 3 (AE1) modulate divalent anion transport and DBDS binding to a second site on band 3 which is activated by lowering the pH (pK 5.0)

Evidence is emerging which indicates that the anion transport activity of band 3 may be regulated. I review the molecular basis for regulation of the anion transport function of band 3 in terms of evidence showing that divalent anion transport involves a slow “hysteretic” transition between two functional states, mediated by interactions between subunits within band 3 oligomers. In addition, I briefly describe recent work from my laboratory where we have discovered a novel manifestation of slow conformational changes in band 3. This involves 4,4′-dibenzamido-2,2′-stilbenedisulfonate (DBDS) binding to a second, proton-activated site distinct from the primary stilbenedisulfonate site. This site is exposed on the outer aspect of band 3 when the pH is lowered (pK 5.0). This is the same pK as the protonation site on band 3 involved in divalent anion–proton co-transport (APCT) [J. Gen. Physiol. 79 (1982) 87]. Significantly, we have found that DBDS binding to this proton-activated site has unusually slow kinetics, and increasing DBDS concentration causes a decrease in the apparent pseudo-first-order rate constant. These results suggest that a slow conformational pre-equilibrium is the rate limiting step in DBDS binding to the proton-activated site on band 3 observed at low pH. Our results support an allosteric two-state model for regulation of divalent anion transport by band 3 oligomers involving a slow conformational transition and interactions between subunits [Biochemistry 31 (1992) 7301].     

Slow transitions between two conformational states of band 3 (AE1) modulate divalent anion transport and DBDS binding to a second site on band 3 which is activated by lowering the pH (pK approximately 5.0)

Evidence is emerging which indicates that the anion transport activity of band 3 may be regulated. I review the molecular basis for regulation of the anion transport function of band 3 in terms of evidence showing that divalent anion transport involves a slow "hysteretic" transition between two functional states, mediated by interactions between subunits within band 3 oligomers. In addition, I briefly describe recent work from my laboratory where we have discovered a novel manifestation of slow conformational changes in band 3. This involves 4,4'-dibenzamido-2,2'-stilbenedisulfonate (DBDS) binding to a second, proton-activated site distinct from the primary stilbenedisulfonate site. This site is exposed on the outer aspect of band 3 when the pH is lowered (pK approximately 5.0). This is the same pK as the protonation site on band 3 involved in divalent anion-proton co-transport (APCT) [J. Gen. Physiol. 79 (1982) 87]. Significantly, we have found that DBDS binding to this proton-activated site has unusually slow kinetics, and increasing DBDS concentration causes a decrease in the apparent pseudo-first-order rate constant. These results suggest that a slow conformational pre-equilibrium is the rate limiting step in DBDS binding to the proton-activated site on band 3 observed at low pH. Our results support an allosteric two-state model for regulation of divalent anion transport by band 3 oligomers involving a slow conformational transition and interactions between subunits [Biochemistry 31 (1992) 7301].     

Switch from a type 2 to a type 1 T helper cell response and cure of established Leishmania major infection in mice is induced by combined therapy with interleukin 12 and Pentostam.

Successful treatment in allergic, autoimmune, and infectious diseases often requires altering the nature of a detrimental immune response mediated by a particular CD4+ T helper (Th) cell subset. While several factors contribute to the development of CD4+ Th1 and Th2 cells, the requirements for switching an established response are not understood. Here we use infection with Leishmania major as a model to investigate those requirements. We report that treatment with interleukin 12 (IL-12), in combination with the antimony-based leishmanicidal drug Pentostam, induces healing in L. major-infected mice and that healing is associated with a switch from a Th2 to a Th1 response. The data suggest that decreasing antigen levels may be required for IL-12 to inhibit a Th2 response and enhance a Th1 response. These observations are important for treatment of nonhealing forms of human leishmaniasis and also demonstrate that in a chronic infectious disease an inappropriate Th2 response can be switched to an effective Th1 response.