Biomedical subjects

Pedro L Vera

Publications and source records attributed to Pedro L Vera.

14 recordsLinked to original sources

PubMed2006

Substance P induces localization of MIF/alpha1-inhibitor-3 complexes to umbrella cells via paracellular transit through the urothelium in the rat bladder.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is released into the intraluminal fluid during bladder inflammation in the rat complexed to alpha1-inhibitor-3 (A1-I3; a rodent proteinase inhibitor in the alpha-macroglobulin family). The location of A1-I3 in the bladder had not been investigated. Therefore, we examined the location of A1-I3 and MIF/A1-I3 complexes in the bladder and changes due to experimental inflammation. METHODS: Anesthetized male rats had bladders removed with no treatment (intact) or were injected with Substance P (SP; s.c.; saline vehicle). After one hour intraluminal fluid was removed, bladder was excised and MIF and A1-I3 levels were determined using ELISA and/or western-blotting. MIF co-immunoprecipitation determined MIF/A1-I3 complexes in the bladder. Bladder sections were immunostained for A1-I3 and MIF/A1-I3. RESULTS: A1-I3 immunostaining was observed in interstitial spaces throughout the bladder (including submucosa) but not urothelium in intact and saline-treated rats. RT-PCR showed that the bladder does not synthesize A1-I3, therefore, A1-I3 in the interstitial space of the bladder must be plasma derived. In SP-treated rats, A1-I3 in the bladder increased and A1-I3 was observed traversing through the urothelium. Umbrella cells that do not show MIF and/or A1-I3 immunostaining in intact or saline-treated rats, showed co-localization of MIF and A1-I3 after SP-treatment. Western blotting demonstrated that in the bladder MIF formed non-covalent interactions and also binds covalently to A1-I3 to form high molecular weight MIF/A1-I3 complexes (170, 130 and 75-kDa, respectively, verified by co-immunoprecipitation). SP-induced inflammation selectively reduced 170-kDa MIF/A1-I3 in the bladder while increasing 170 and 130-kDa MIF/A1-I3 in the intraluminal fluid. CONCLUSION: A1-I3 and MIF/A1-I3 complexes are resident in bladder interstitium. During SP-induced inflammation, MIF/A1-I3 complexes are released from the bladder into the lumen. Binding of MIF/A1-I3 complexes to urothelial cells during inflammation suggests these complexes participate in the inflammatory reaction through activation of receptors for MIF and/or for A1-I3.

Acute-Phase Proteins↗

PubMed2006

Macrophage migration inhibitory factor is increased in the urine of patients with urinary tract infection: macrophage migration inhibitory factor-protein complexes in human urine.

PURPOSE: MIF is a proinflammatory cytokine present in preformed stores in human urothelium. In animal models of bladder inflammation, including bacterial cystitis, MIF is up-regulated in the bladder and released from the bladder as a high molecular weight complex. We compared urine MIF amounts in patients with UTI to that in patients without UTI, and we examined and identified MIF-protein complexes in urine. MATERIALS AND METHODS: Using enzyme-linked immunosorbent assay we compared MIF levels in the urine of 14 patients with UTI to levels in 16 controls with no UTI. Western blotting under native, denaturing and reducing conditions was done to examine MIF complexes found in urine. Mass spectrometry identified MIF associated proteins in urine, while co-immunoprecipitation confirmed the associations. RESULTS: Mean urine MIF amounts +/- SEM determined by enzyme-linked immunosorbent assay were significantly greater in 14 patients with UTI compared to that in 16 controls (1.96 +/- 0.40 vs 0.59 +/- 0.09 ng/mg creatinine, p <0.01). Western blotting under denaturing conditions showed several high molecular weight complexes (100 to 165 kDa) that increased in UTI urine as well as typical, monomeric MIF (12 kDa). Mass spectrometry identified associated MIF proteins, including ceruloplasmin, albumin and uromodulin. Co-immunoprecipitation confirmed mass spectrometry findings and also identified MIF interaction with alpha-2-macroglobulin. CONCLUSIONS: Increased urine MIF amounts in patients with bacterial cystitis support our experimental evidence showing a role for MIF in pelvic visceral inflammation. The novel finding of an association of MIF with other urine proteins suggest that the physiologically relevant form of MIF may be an MIF-protein complex.

Bacterial Infections↗

PubMed2005

Further evidence for increased macrophage migration inhibitory factor expression in prostate cancer.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a cytokine associated with prostate cancer, based on histologic evidence and circulating (serum) levels. Recent studies from another laboratory failed to document these results. This study's aims were to extend and confirm our previous data, as well as to define possible mechanisms for the discrepant results. Additional aims were to examine MIF expression, as well as the location of MIF's receptor, CD74, in human prostatic adenocarcinoma compared to matched benign prostate. METHODS: MIF amounts were determined in random serum samples remaining following routine PSA screening by ELISA. Native, denaturing and reducing polyacrylamide gels and Western blot analyses determined the MIF form in serum. Prostate tissue arrays were processed for MIF in situ hybridization and immunohistochemistry for MIF and CD74. MIF released into culture medium from normal epithelial, LNCaP and PC-3 cells was detected by Western blot analysis. RESULTS: Median serum MIF amounts were significantly elevated in prostate cancer patients (5.87 +/- 3.91 ng/ml; +/- interquartile range; n = 115) compared with patients with no documented diagnosis of prostate cancer (2.19 +/- 2.65 ng/ml; n = 158). ELISA diluent reagents that included bovine serum albumin (BSA) significantly reduced MIF serum detection (p < 0.01). MIF mRNA was localized to prostatic epithelium in all samples, but cancer showed statistically greater MIF expression. MIF and its receptor (CD74) were localized to prostatic epithelium. Increased secreted MIF was detected in culture medium from prostate cancer cell lines (LNCaP and PC-3). CONCLUSION: Increased serum MIF was associated with prostate cancer. Diluent reagents that included BSA resulted in MIF serum immunoassay interference. In addition, significant amounts of complexed MIF (180 kDa under denaturing conditions by Western blot) found in the serum do not bind to the MIF capture antibody. Increased MIF mRNA expression was observed in prostatic adenocarcinoma compared to benign tissue from matched samples, supporting our earlier finding of increased MIF gene expression in prostate cancer.

Adenocarcinoma↗

PubMed2005

Substance P induced changes in CD74 and CD44 in the rat bladder.

PURPOSE: Substance P (SP) induces rat bladder inflammation along with release of the proinflammatory cytokine, macrophage migration inhibitory factor (MIF). To describe the mechanism of MIF action we examined changes in the amount of CD74 (membrane receptor for MIF), CD44 and phospho-(p-ERK)1/2 in the bladder. MATERIALS AND METHODS: In anesthetized rats the bladder was isolated by cutting the ureters and urine was replaced by saline as intraluminal fluid (ILF). One hour after subcutaneous SP (40 mug/kg) or saline administration the ILF and bladder were collected. Bladder tissue was analyzed for CD74 and CD44 by immunohistochemistry. Western blot analysis determined the relative amounts of bladder tissue MIF, CD74, CD44 and p-ERK1/2. ILF immunoprecipitation followed by Western blot analysis was performed to identify an association of MIF with CD74 and/or CD44. RESULTS: SP induced significant MIF release from the bladder and increased CD74 and CD44 bladder immunostaining. SP treatment increased the total amount of bladder CD74 protein and mRNA, intracellular domain CD44, p-ERK1/2 and soluble CD44 in the ILF. Finally, MIF was found to be associated with soluble CD44 in the ILF. CONCLUSIONS: CD74 is present in the rat urothelium. SP increases CD74 and intracellular domain CD44 in the bladder, while stimulating the release of soluble CD44 and MIF into the ILF. MIF interacts with soluble CD44 in the ILF and it is available to bind with CD74 in the bladder to exert proinflammatory effects. Therefore, a mechanistic model is emerging to explain the proinflammatory effects of MIF in this acute model of bladder inflammation. Possible clinical implications are discussed.

Animals↗

PubMed2005

Macrophage migration inhibitory factor is released as a complex with alpha1-inhibitor-3 in the intraluminal fluid during bladder inflammation in the rat.

PURPOSE: Macrophage migration inhibitory factor (MIF) is released into intraluminal fluid (ILF) during bladder inflammation in the rat. We investigated the forms of MIF that are released. We examined MIF release after subcutaneous substance P (SP) or intravesical capsaicin and studied proteins associated with excreted MIF in ILF. MATERIALS AND METHODS: Anesthetized male rats with the bladder isolated from the kidneys were injected with SP subcutaneously (saline vehicle) or with intravesical capsaicin (vehicle, 0.1 mM and 1 mM). After 1 hour the ILF was removed and MIF levels were determined using enzyme-linked immunosorbent assay or Western blotting procedures under native, nonreducing and reducing conditions. Mass spectrometry was used to identify proteins associated with MIF in ILF and results were verified by immunoprecipitation. RESULTS: SP and intravesical capsaicin increased the total amount of MIF in ILF. MIF was found in high molecular weight complexes that resolved into 2 bands under nonreducing conditions. SP and capsaicin differentially increased the MIF bands. Mass spectrometry determined that MIF was complexed with acute phase proteins. MIF immunoprecipitation followed by Western blotting confirmed that MIF was complexed to alpha1-inhibitor-3. CONCLUSIONS: MIF is complexed with alpha1-inhibitor-3, a member of the alpha-2-macroglobulin proteinase inhibitor family, in the rat. Although SP and capsaicin increased the total amount of MIF detected by enzyme-linked immunosorbent assay in ILF, the patterns of MIF complexes elicited by these 2 treatments were different. These findings suggest that in association with other proteins MIF forms part of a complex elicited by bladder inflammation.

Acute-Phase Proteins↗

PubMed2004

Inhibition of macrophage migration inhibitory factor decreases proliferation and cytokine expression in bladder cancer cells.

BACKGROUND: The importance of various inflammatory cytokines in maintaining tumor cell growth and viability is well established. Increased expression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) has previously been associated with various types of adenocarcinoma. METHODS: MIF IHC was used to localize MIF in human bladder tissue. ELISA and Western blot analysis determined the synthesis and secretion of MIF by human bladder transitional cell carcinoma cells. The effects of MIF inhibitors (high molecular weight hyaluronate (HA), anti-MIF antibody or MIF anti-sense) on cell growth and cytokine expression were analyzed. RESULTS: Human bladder cancer cells (HT-1376) secrete detectable amounts of MIF protein. Treatment with HA, anti-MIF antibody and MIF anti-sense reduced HT-1376 cell proliferation, MIF protein secretion, MIF gene expression and secreted inflammatory cytokines. Our evidence suggests MIF interacts with the invariant chain, CD74 and the major cell surface receptor for HA, CD44. CONCLUSIONS: This study is the first to report MIF expression in the human bladder and these findings support a role for MIF in tumor cell proliferation. Since MIF participates in the inflammatory response and bladder cancer is associated with chronic inflammatory conditions, these new findings suggest that neutralizing bladder tumor MIF may serve as a novel therapeutic treatment for bladder carcinoma.

Antigens, CD↗

PubMed2004

Macrophage migration inhibitory factor is upregulated in an endotoxin-induced model of bladder inflammation in rats.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine found in epithelial cells as preformed stores, such that MIF release can activate innate immune responses. Our identification of MIF stores in the urothelium suggests that MIF may function in the bladder's initial response to infectious stimuli, such as lipopolysaccharide (LPS). To test this hypothesis, we observed changes in MIF, cyclooxygenase-2 (COX-2) and c-fos in the bladder, L6-S1 spinal cord, dorsal root ganglion (DRG), and major pelvic ganglion (MPG) and MIF changes in the prostate following intravesical LPS. Intravesical LPS induced bladder edema and leukocyte infiltration, as well as increased MIF protein and mRNA in the bladder and lumbosacral spinal cord. Expression of immediate-early gene c-fos, a transcription factor used as a marker of neuronal activation, increased in the L6-S1 spinal cord and L6-S1 DRG of rats that received LPS. We conclude that significant increases in bladder MIF expression and protein in response to intravesical LPS may represent part of this organ's initial innate immune response. In addition, MIF upregulation may represent a neural response to visceral inflammation. Finally, changes in prostate MIF content after intravesical LPS suggest that MIF may be involved in viscerovisceral interactions associated with chronic pelvic pain syndromes.

Animals↗

PubMed2004

Substance P induced release of macrophage migration inhibitory factor from rat bladder epithelium.

PURPOSE: Substance P (SP), a neuropeptide mediator of neurogenic inflammation, induces vasodilatation, plasma extravasation and hypersensitivity in the bladder. SP induced inflammation is enhanced and maintained by the release of additional mediators. The rat urothelium contains pre-formed macrophage migration inhibitory factor (MIF), a known proinflammatory cytokine, suggesting that it may mediate bladder neurogenic inflammation. We documented the time course of SP effects on rat bladder inflammation. MATERIALS AND METHODS: Subcutaneous SP administration induced neurogenic inflammation. The bladder, urine and serum were removed 15, 30, 60 and 120 minutes following treatment, and changes in MIF, nerve growth factor (NGF), c-fos and cox-2 were determined. RESULTS: SP induced significant MIF and NGF release from the bladder following 30 minutes of exposure. cox-2 protein was detected at significant levels following 60 minutes of SP exposure. Basal c-fos protein could be detected in control bladders with significant increases following 60 minutes of SP exposure. Histological examination of bladder tissue showed increased edema in SP treated bladders. CONCLUSIONS: SP stimulated early release of urothelial MIF as well as increased MIF gene expression in this model of neurogenic inflammation. SP also increased expression of the proinflammatory mediator NGF. In addition, increases in cox-2 enzyme and c-fos transcription factor were noted. The early release of MIF suggests that it is an immediate proinflammatory regulator in the bladder and it establishes MIF as candidate proinflammatory mediator of SP induced neurogenic inflammation. These data continue to support our hypothesis that MIF is a new target for intervention in bladder inflammation.

Animals↗

PubMed2004

Inflammation of the rat prostate evokes release of macrophage migration inhibitory factor in the bladder: evidence for a viscerovisceral reflex.

PURPOSE: Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is found in preformed stores in bladder epithelium. We examined the effects of prostatic inflammation on micturition frequency, bladder histology and bladder MIF content as a model in which to study viscerovisceral reflexes mediating pelvic visceral inflammation. MATERIALS AND METHODS: Cystometry was performed in urethane anesthetized male rats. Formalin or saline was injected into the ventral lobe of the prostate to induce inflammation. Cystometry continued 1 hour after injection. The bladder, ventral lobes of the prostates and lumbosacral spinal cord were then removed, and protein levels and gene expression of MIF, cyclooxygenase-2 (COX-2) and nerve growth factor (NGF) were examined. Edema was verified histologically in the bladder and prostate. RESULTS: Intraprostatic formalin produced almost immediate bladder hyperreflexia, which was maintained during the observation period. Bladder edema was noted during histological examination. Bladder MIF protein amounts decreased, while COX-2 and NGF increased after prostatic injection. Bladder MIF, COX-2 and NGF mRNA increased. In the lumbosacral spinal cord protein and mRNA amounts increased for all factors examined in animals that received intraprostatic formalin. No changes were observed in the cervical cord. Rats injected with formalin mixed with dye showed restriction of the dye to the prostate. CONCLUSIONS: A viscerovisceral reflex in the rat, probably mediated by the lumbosacral spinal cord, produced bladder hyperreflexia and bladder edema, and evoked MIF release from the bladder and the induction of other inflammatory mediators. This supports our hypothesis that MIF is involved in neurogenic inflammation in the pelvic viscera and it may represent an interesting therapeutic target.

Animals↗

PubMed2004

Intraluminal antibodies to macrophage migration inhibitory factor decrease substance P induced inflammatory changes in the rat bladder and prostate.

PURPOSE: Noxious stimuli induce substance P (SP) secretion from nerve terminals, resulting in plasma extravasation, edema and hyperalgesia, commonly referred to as neurogenic inflammation. Since SP is a short-lived molecule, additional proinflammatory mediators maintain continued inflammation. The bladder contains stores of preformed macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, which is released into the lumen in response to SP. MIF may act in an amplifying manner to maintain or increase inflammation. Inducing inflammatory changes with SP, while sequestering released luminal MIF with an antibody, tested this hypothesis. MATERIALS AND METHODS: In anesthetized rats the ureters were cut to isolate the bladder and the bladder contents were replaced with saline or antiMIF antibody (5 or 15 microg/ml), immediately followed by systemic SP or saline. Changes in the expression of inflammatory cytokines, and histological changes in the bladder and prostate were evaluated 1 hour later. RESULTS: : Targeted array analysis identified increases in proinflammatory gene expression in the bladder and prostate as a result of SP. SP induced changes in MIF, cyclooxygenase-2, nerve growth factor, c-fos and edema were decreased by intraluminal anti-MIF. CONCLUSIONS: SP increased MIF amounts in the bladder lumen. Sequestering luminal MIF with an antiMIF antibody decreased SP induced inflammatory changes in the bladder and prostate, suggesting that MIF is involved in acute pelvic visceral neurogenic inflammation. These data indicate that MIF released from the bladder sustains or amplifies SP induced inflammation, a possibility that agrees with known MIF proinflammatory functions. These data continue to support our hypothesis that MIF is a new target for intervention in pelvic viscera inflammation.

Animals↗

PubMed2003

Anatomical location of macrophage migration inhibitory factor in urogenital tissues, peripheral ganglia and lumbosacral spinal cord of the rat.

BACKGROUND: Previous work suggested that macrophage migration inhibitory factor (MIF) may be involved in bladder inflammation. Therefore, the location of MIF was determined immunohistochemically in the bladder, prostate, major pelvic ganglia, sympathetic chain, the L6-S1 dorsal root ganglia (DRG) and the lumbosacral spinal cord of the rat. RESULTS: In the pelvic organs, MIF immunostaining was prominent in the epithelia. MIF was widely present in neurons in the MPG and the sympathetic chain. Some of those neurons also co-localized tyrosine hydroxylase (TH). In the DRGs, some of the neurons that stained for MIF also stained for Substance P. In the lumbosacral spinal cord, MIF immunostaining was observed in the white mater, the dorsal horn, the intermediolateral region and in the area around the central canal. Many cells were intensely stained for MIF and glial fibrillary acidic protein (GFAP) suggesting they were glial cells. However, some cells in the lumbosacral dorsal horn were MIF positive, GFAP negative cells suggestive of neurons. CONCLUSIONS: Therefore, MIF, a pro-inflammatory cytokine, is localized to pelvic organs and also in neurons of the peripheral and central nervous tissues that innervate those organs. Changes in MIF's expression at the end organ and at peripheral and central nervous system sites suggest that MIF is involved in pelvic viscera inflammation and may act at several levels to promote inflammatory changes.

Animals↗

PubMed2003

Hydrochloric acid induced changes in macrophage migration inhibitory factor in the bladder, peripheral and central nervous system of the rat.

PURPOSE: We established the presence of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) in the bladder and in nervous system structures innervating the bladder, and evaluated changes in MIF and cyclooxygenase-2 (COX-2) protein levels and expression following chemical cystitis. MATERIALS AND METHODS: Male Sprague-Dawley rats were anesthetized and a catheter was introduced into the bladder dome. Cystitis was induced by infusing 0.4 N HCl into the bladder. Control rats received a similar volume of saline. Two hours later the bladder, major pelvic ganglia (MPG), L6/S1 dorsal root ganglia (DRG) and L6/S1 spinal cord were removed and assayed for MIF and COX-2 protein, and mRNA using Western blot and quantitative reverse transcriptase-polymerase chain reaction techniques. RESULTS: Immunohistochemistry showed MIF located mainly in the urothelium of saline treated rats. Instillation of HCl into the bladder resulted in marked epithelial denudation, moderate edema and vasodilatation in the submucosa. MIF protein levels decreased but MIF mRNA expression remained unchanged in bladders treated with HCl compared with controls. However, MIF protein and mRNA levels increased in the MPG, L6/S1 DRG and L6/S1 spinal cord of HCl treated animals. COX-2 protein was not detected in the bladder, DRG or MPG of saline-treated rats. However, a small amount was present in the L6/S1 cord. On the other hand, HCl treated rats showed marked increases in COX-2 protein levels in all tissues examined. Similarly although cox-2 mRNA was constitutively expressed in all tissues examined, expression increased following HCl treatment. CONCLUSIONS: Chemical cystitis induced by intravesical HCl in rats increases the protein levels and mRNA expression of MIF and COX-2 in central and peripheral nervous system tissues that are involved in innervating the bladder. This finding suggests that MIF may be involved in bladder inflammation and may have a role in the peripheral and central nervous system pathways that regulate bladder reflexes in response to bladder inflammation.

Animals↗

PubMed2002

Separate urinary bladder and prostate neurons in the central nervous system of the rat: simultaneous labeling with two immunohistochemically distinguishable pseudorabies viruses.

BACKGROUND: This work examines the central nervous system distribution of virus-labeled neurons from the rat urinary bladder and the prostate simultaneously within the same tissue sections. Two immunohistochemically distinct pseudorabies virus strains were simultaneously injected into male Sprague Dawley rats (approximately 280 gm). One virus was injected into the bladder and the other into the prostate. After incubation intervals of 2.25, 2.5, 2.75, 3 and 4 days, sections from the spinal cord and brain were processed immunohistochemically to detect cells, within a single section, which were labeled separately by each virus or were labeled by both viruses. RESULTS: Each strain of virus labeled a separate population of neurons and some neurons were labeled by both strains. The majority of neurons labeled by virus from the urinary bladder were found in the L6-S1 spinal cord segments within the dorsal gray commissure, the intermediolateral area and the superficial dorsal horn. Neurons labeled by virus from the prostate were mainly found in the L1-L2 spinal cord segments in the dorsal gray commissure and the intermediolateral areas. Double-labeled interneurons in L1-L2 were mainly located in the intermediolateral area. In L6-S1 they were divided between the dorsal gray commissure and the intermediolateral area. CONCLUSIONS: Spinal neurons innervating the bladder are clearly separate and different from those innervating the prostate. This difference also persists in the brain. In disagreement with previous reports, no direct anatomical evidence of parasympathetic innervation of the prostate was observed.

Animals↗

PubMed2002

Central effects of clozapine in regulating micturition in anesthetized rats.

BACKGROUND: We previously showed that systemic administration of the atypical neuroleptic clozapine in the rat altered a number of urodynamic variables and inhibited the external urethral sphincter. Since clozapine acts at several receptor types both at the periphery and the central nervous system, the site of action remained uncertain. Therefore, the purpose of this study was to determine the effects of central administration of clozapine on the bladder and the external urethral sphincter during cystometry and to examine differences in spinal versus supraspinal administration. We extended our observations by delivering clozapine centrally in anesthetized rats instrumented with either an intrathecal (L6-S1 spinal segment) or an intracerebroventricular (lateral ventricle) catheter. RESULTS: Clozapine decreased micturition volume and increased residual volume possibly by acting at a supraspinal site. Expulsion time and amplitude of the high frequency oscillations were reduced by clozapine possibly by acting at a spinal site. Bladder capacity was increased after central clozapine but probably due to a peripheral effect. Clozapine acting at spinal and supraspinal sites increased pressure threshold. Contraction time and peak pressure were not affected by clozapine. The EMG from the external urethral sphincter was also reduced following clozapine centrally and suggests a spinal and a supraspinal site of action. CONCLUSIONS: The results from the present study suggest that spinal and supraspinal central sites mediate clozapine's action in inhibiting expulsion parameters and the external urethral sphincter of the rat. Therefore, the reduction in the voiding efficiency observed after clozapine appears to be mediated by spinal and supraspinal sites.

Animals↗