Biomedical subjects
A E Anderson
Publications and source records attributed to A E Anderson.
Kainate mediates nuclear factor-kappa B activation in hippocampus via phosphatidylinositol-3 kinase and extracellular signal-regulated protein kinase.
The transcription factor nuclear factor-kappa B (NF-kappaB) is an inducible regulator of genes that plays a crucial role in the nervous system. Glutamate receptor stimulation is one well-described mechanism for NF-kappaB activation. In the studies presented here we used the glutamate analog, kainate to investigate the signaling mechanisms that couple to NF-kappaB activation in hippocampus. Kainate (250 nM) application to hippocampal slices elicited a time-dependent increase in nuclear NF-kappaB levels in areas CA3 and CA1, but not dentate, compared with controls. Further analysis focused on hippocampal area CA3, revealed increased NF-kappaB DNA binding activity in response to kainate stimulation. Supershift electrophoretic mobility shift assay indicated that the kainate-mediated NF-kappaB complex binding DNA was composed of p65, p50, and c-Rel subunits. Through inhibition studies we found that extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) couple to basal and kainate-mediated NF-kappaB DNA binding activity in area CA3. Kainate elicited decreased total and increased phospho-inhibitor kappa B alpha (IkappaBalpha), suggesting that kainate-mediated activation of NF-kappaB is via the classical IkappaB kinase pathway. Interestingly, inhibition of ERK but not PI3K blocked the kainate-mediated increase in phospho-IkappaBalpha. Thus, our findings support a role for the ERK and PI3K pathways in kainate-mediated NF-kappaB activation in hippocampal area CA3, but these kinases may target the NF-kappaB pathway at different loci.
Leitmotifs in the biochemistry of LTP induction: amplification, integration and coordination.
Hippocampal long-term potentiation (LTP) is a robust and long-lasting form of synaptic plasticity that is the leading candidate for a cellular mechanism contributing to mammalian learning and memory. Investigations over the past decade have revealed that the biochemistry of LTP induction involves mechanisms of great subtlety and complexity. This review highlights themes that have emerged as a result of our increased knowledge of the signal transduction pathways involved in the induction of NMDA receptor-dependent LTP in area CA1 of the hippocampus. Among these themes are signal amplification, signal integration and signal coordination. Here we use these themes as an organizing context for reviewing the profusion of signaling mechanisms involved in the induction of LTP.
Loss of the potassium channel beta-subunit gene, KCNAB2, is associated with epilepsy in patients with 1p36 deletion syndrome.
PURPOSE: Clinical features associated with chromosome 1p36 deletion include characteristic craniofacial abnormalities, mental retardation, and epilepsy. The presence and severity of specific phenotypic features are likely to be correlated with loss of a distinct complement of genes in each patient. We hypothesize that hemizygous deletion of one, or a few, critical gene(s) controlling neuronal excitability is associated with the epilepsy phenotype. Because ion channels are important determinants of seizure susceptibility and the voltage-gated K(+) channel beta-subunit gene, KCNAB2, has been localized to 1p36, we propose that deletion of this gene may be associated with the epilepsy phenotype. METHODS: Twenty-four patients were evaluated by fluorescence in situ hybridization with a probe containing KCNAB2. Clinical details were obtained by neurologic examination and EEG. RESULTS: Nine patients are deleted for the KCNAB2 locus, and eight (89%) of these have epilepsy or epileptiform activity on EEG. The majority of patients have a severe seizure phenotype, including infantile spasms. In contrast, of those not deleted for KCNAB2, only 27% have chronic seizures, and none had infantile spasms. CONCLUSIONS: Lack of the beta subunit would be predicted to reduce K(+) channel-mediated membrane repolarization and increase neuronal excitability, suggesting a possible relation between loss of this gene and the development of seizures. Because some patients with seizures were not deleted for KCNAB2, there may be additional genes within 1p36 that contribute to epilepsy in this syndrome. Hemizygosity of this gene in a majority of monosomy 1p36 syndrome patients with epilepsy suggests that haploinsufficiency for KCNAB2 is a significant risk factor for epilepsy.
A report of intracranial Rosai-Dorfman disease with literature review.
Rosai-Dorfman disease (sinus histiocytosis with massive lymphadenopathy), is a rare benign histiocytic proliferative disorder. Over 650 cases have been reported since 1969. To the best of our knowledge, there have been only 31 cases with central nervous system involvement reported in the literature. Intracranial disease usually presents clinically and radiologically as a "meningioma". It can be misdiagnosed as a nonspecific inflammatory process because of the atypical histologic features of Rosai-Dorfman disease occurring in a non-nodal location. Familiarity with such atypical histologic features and appropriate use of immunohistochemical stains is required for a definitive diagnosis of central nervous system Rosai-Dorfman disease. We report such an intracranial lesion with other extranodal sites of involvement with a 5-year follow up and a review of previously reported cases. Ann Diagn Pathol 5:96-102, 2001.
Laparoscopic ureteral reconstruction with small intestinal submucosa.
PURPOSE: To evaluate the feasibility of laparoscopic ureteral reconstruction with small intestinal submucosa (SIS) in the pig ureter. MATERIALS AND METHODS: Eight female pigs weighing between 25 and 30 kg were enrolled. After anesthesia was administered, a double-pigtail stent was inserted, the animals were moved to a lateral decubitus position, pneumoperitoneum was established, and three 10-mm ports were positioned. The ureter was opened longitudinally for 7 cm, and two thirds of the periphery of the upper third of the left ureter was excised. The SIS was anastomosed to the upper and distal ureteral segments with chromic 4-0 sutures. The double-pigtail stent was removed 6 weeks after the initial procedure, and retrograde pyelography was performed a week later to confirm the viability of the pelvicaliceal system. RESULTS: The average duration of the procedures was 210 minutes (range 125-250 minutes). All animals survived the entire follow-up period of 7 weeks. Retrograde pyelography revealed a patent ureteral lumen, and no obstructive phenomena were observed. Histologically, the SIS-regenerated ureteral segments were remarkably similar to normal porcine ureters and were indistinguishable from neighboring tissue. CONCLUSION: Laparoscopic ureteral reconstruction with SIS proved to be effective and technically feasible. The SIS seems to be an effective biodegradable scaffold, facilitating regeneration of host tissue.
Antigenic characterization of medullary carcinoma of the breast: HLA-DR expression in lymph node positive cases.
Medullary carcinoma of the breast has attracted attention because of its relatively good prognosis, in spite of its high cytologic grade. It has, by definition, a consistent, florid tumor infiltrating lymphocyte (TIL) population, probably the result of cytotoxic T-lymphocytes recognizing tumor cells in an HLA-DR-restricted manner. HLA-DR tends to be more highly expressed on primary medullary carcinoma cells than on ductal carcinoma cells; however, the MHC-class II antigenicity of the tumor cells themselves has not been analyzed extensively, and as yet there has been no comparative study of HLA-DR expression in medullary and ductal carcinomas metastatic to lymph nodes. Eleven cases of medullary carcinoma and 15 cases of ductal carcinoma, primaries, and respective lymph node metastases were analyzed by immunoperoxidase staining for HLA-DR and lymphocytes antigens. Polymerase chain reaction (PCR) analysis to identify HLA-DR subtypes from the paraffin blocks was performed on selected cases of primaries and nodal metastases of both tumor types. Immunoperoxidase staining for HLA-DR antigen revealed a marked difference in antigen expression between medullary and ductal carcinomas. In the medullary carcinomas, the mean percentage of cells staining for HLA-DR was 74.5% in the primary tumors and 67.3% in the nodal metastases. For the ductal carcinomas, the mean percentage of cells staining was 17.7% in the primaries and 7% in the metastases. There was a tendency for the level of HLA-DR expression to remain high in medullary carcinoma metastatic to nodes, whereas whatever HLA-DR was present within ductal primaries tended to diminish when cells metastasized to regional nodes. PCR analysis of the HLA-DR within the two tumor types revealed no emerging subtype or variant that could be associated with either the medullary or the ductal carcinomas. Medullary carcinoma cells express much greater quantities of HLA-DR, on the whole, than ductal carcinomas. Expression of HLA-DR is retained on medullary carcinoma cells that have spread to lymph nodes, whereas the smaller quantities of HLA-DR present within ductal primaries tend to diminish even further when the tumor cells are found in lymph nodes. No discernible HLA-DR mutations or predominant subtypes emerged on PCR analysis, and the authors therefore conclude that it is the quantity and not the quality of HLA-DR expression in medullary carcinoma that maintains the characteristic TIL infiltrate, not seen in ductal carcinomas.
Sodium bisulfite analysis of the methylation status of DNA from small portions of paraffin slides.
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Ureteral reconstruction: small intestine submucosa for the management of strictures and defects of the upper third of the ureter.
PURPOSE: We evaluated the effectiveness of small intestine submucosa in ureteral reconstruction. MATERIALS AND METHODS: We report an experimental study in 6 female pigs weighing between 50 and 60 pounds. The animals were anesthetized, midline laparotomy was performed and two-thirds of the diameter of the upper third of the left ureter were incised parallel to the ureteral axis, leaving intact only a third of the ureteral wall for a segment of 7 cm. A 5Fr double-J*; stent was positioned to secure patency at all times. The created gap was then bridged with an small intestine submucosa patch in a cylindrical format, which was subsequently sutured to the proximal and distal ureteral segment. The right ureter served as our control and simple intubated Davis ureterotomy was performed. RESULTS: All animals survived the whole followup of 7 weeks. Histologically there was evidence of epithelial regeneration along the segments reconstructed with small intestine submucosa, supported by a well vascularized collagen and smooth muscle background. There was no evidence of foreign body reaction to the graft material. In vivo patency was confirmed by retrograde pyelography in the bridged ureters 7 weeks after the initial procedure. All the ureters without an small intestine submucosa bridge had ureteral stenosis without evidence of epithelial regeneration. CONCLUSIONS: The use of small intestine submucosa is a novel, effective material for the scaffolding of ureteral defects and/or strictures of the upper ureteral segment in the pig model.
Chronic expansion of the renal pelvis: a new method for reconstruction of upper ureteral defects.
OBJECTIVES: To evaluate whether the dilated renal pelvis can be used as an autologous source for the surgical reconstruction of upper ureteral defects or strictures. METHODS: In 7 female pigs, the renal pelvis was expanded by a percutaneously placed Council balloon catheter. Every other day for 4 weeks, the renal pelvis was progressively dilated with a bolus injection of saline and contrast medium, which allowed expansion of the renal pelvis to 70 to 75 mL. Four to six weeks after the initial intervention, 5 to 7 cm of the proximal ureter was resected in an open operation and replaced with a tubularized spiral flap made from the expanded renal pelvis. Three weeks later, the animals were killed, and the area of manipulation was resected for pathologic evaluation. RESULTS: All animals reached the desired expansion of the renal pelvis, and in all cases, the spiral flap was fashioned and anastomosed to the distal ureteral segment, bridging the initial defect (mean length 7 cm). Two animals died from sepsis, 4 and 6 days after the spiral flap reconstruction. A viable ureteral lumen with a patent anastomosis and a functioning pelvocaliceal unit was observed on intravenous urography in all animals. The main histologic findings were a chronic inflammatory process with concomitant mucinous metaplasia and reactive atypia of the tubules. CONCLUSIONS: The use of a balloon expander in the renal pelvis is a safe and effective technique for producing native tissue for the reconstruction of defects or strictures of the upper ureteral segment.
Kv4.2 phosphorylation by cyclic AMP-dependent protein kinase.
Recent evidence suggests that K(+) channels composed of Kv4.2 alpha-subunits underlie a transient current in hippocampal CA1 neurons and ventricular myocytes, and activation of the cAMP second messenger cascade has been shown to modulate this transient current. We determined if Kv4.2 alpha-subunits were directly phosphorylated by cAMP-dependent protein kinase (PKA). The intracellular domains of the amino and carboxyl termini of Kv4.2 were expressed as glutathione S-transferase fusion protein constructs; we observed that both of these fusion proteins were substrates for PKA in vitro. By using phosphopeptide mapping and amino acid sequencing, we identified PKA phosphorylation sites on the amino- and carboxyl-terminal fusion proteins corresponding to Thr(38) and Ser(552), respectively, within the Kv4.2 sequence. Kinetic characterization of the PKA sites demonstrated phosphorylation kinetics comparable to Kemptide. To evaluate PKA site phosphorylation in situ, phospho-selective antisera for each of the sites were generated. By using COS-7 cells expressing an EGFP-Kv4.2 fusion protein, we observed that stimulation of the endogenous PKA cascade resulted in an increase in phosphorylation of Thr(38) and Ser(552) within Kv4.2 in the intact cell. We also observed modulation of PKA phosphorylation at these sites within Kv4.2 in hippocampal area CA1. These results provide insight into likely sites of regulation of Kv4.2 by PKA.
The A-type potassium channel Kv4.2 is a substrate for the mitogen-activated protein kinase ERK.
The mitogen-activated protein kinase ERK has recently become a focus of studies of synaptic plasticity and learning and memory. Due to the prominent role of potassium channels in regulating the electrical properties of membranes, modulation of these channels by ERK could play an important role in mediating learning-related synaptic plasticity in the CNS. Kv4.2 is a Shal-type potassium channel that passes an A-type current and is localized to dendrites and cell bodies in the hippocampus. The sequence of Kv4.2 contains several consensus sites for ERK phosphorylation. In the present studies, we tested the hypothesis that Kv4.2 is an ERK substrate. We determined that the Kv4.2 C-terminal cytoplasmic domain is an effective ERK2 substrate, and that it is phosphorylated at three sites: Thr(602), Thr(607), and Ser(616). We used this information to develop antibodies that recognize Kv4.2 phosphorylated by ERK2. One of our phospho-site-selective antibodies was generated using a triply phosphorylated peptide as the antigen. We determined that this antibody recognizes ERK-phosphorylated Kv4.2 in COS-7 cells transfected with Kv4.2 and native ERK-phosphorylated Kv4.2 in the rat hippocampus. These observations indicate that Kv4.2 is a substrate for ERK in vitro and in vivo, and suggest that ERK may regulate potassium-channel function by direct phosphorylation of the pore-forming alpha subunit.
Insights into the tetanus toxin model of early-onset epilepsy from long-term video monitoring during anticonvulsant therapy.
Video monitoring studies were undertaken to determine if the anticonvulsant, carbamazepine (CBZ), could prevent seizures in infant rats that had been intrahippocampally injected with tetanus toxin (TNTX). In control rats, seizure frequency peaked 5-6 days after injection and rapidly declined by postinjection day 9. Twice-daily CBZ treatments dramatically suppressed behavioral seizures for 7 days. However, despite increasing the dosage of CBZ, rats experienced more behavioral seizures during the second week after TNTX injection. Paradoxically, tetanus-toxin-injected control rats had very few seizures at this time. Results not only suggest that this TNTX model may be useful in screening drugs for treating intractable focal epilepsy of infancy but also provide some insight into the processes that may contribute to the rapid decline in behavioral seizure frequency that occurs during the acute phase of epileptogenesis in this model.
A chronic focal epilepsy with mossy fiber sprouting follows recurrent seizures induced by intrahippocampal tetanus toxin injection in infant rats.
Studies were conducted to characterize a chronic epileptic condition that follows recurrent seizures induced by intrahippocampal tetanus toxin injection in infancy. Wistar rat pups received a single injection of tetanus toxin in the right CA3 region on postnatal day 10. Animals were monitored for epileptiform activity by video electroencephalographic or visual observation during the following three to five days. Repeat evaluation six months later demonstrated interictal discharges in 79% (11 of 14) and electrographic seizures in 42% (six of 14) of adult rats with tetanus toxin-induced seizures in infancy. Five of the animals had interictal activity which occurred focally in either the left (n = 2) or right (n = 3) hippocampus. One animal had focal interictal activity independently in these regions and in the left and right cortical regions. The remaining five animals had interictal activity in the hippocampus and synchronously in the ipsilateral cortex or the contralateral hippocampus. Electrographic seizures were focal (nine of 14) or bilateral (five of 14) in onset. The behaviors that accompanied these seizures were quite variable. Clonic face and forelimb movements were observed in some animals. However, a significant portion of rats had electrographic seizures with no associated behavioral change. Timm staining was performed on hippocampal sections from experimental and control animals. There was a significantly greater Timm score (aberrant Timm granules) in the inner molecular layer of the dentate gyrus in tetanus toxin-treated rats than in control rats. Our findings suggest that intrahippocampal tetanus toxin injection in infant rats results in a chronic focal epilepsy that persists for at least six months and is associated with aberrant mossy fiber sprouting in the dentate gyrus. The model described here contributes significantly to the evidence for chronic effects of recurrent seizures in early life, and provides a model for investigation of the molecular and cellular events that contribute to the development of chronic epilepsy.
Failed endopyelotomy: low expression of TGF beta regardless of the presence or absence of crossing vessels.
BACKGROUND AND OBJECTIVE: Endopyelotomy relies on Davis' intubated ureterotomy principle of healing by secondary intention and smooth-muscle regeneration. Approximately 15% of endopyelotomies fail, and the restrictured segment almost always shows evidence of reactive fibrosis with little smooth-muscle regeneration. Previous data suggests that an elevation of TGF beta in obstructed ureteropelvic junctures may be necessary for successful tissue repair following endopyelotomy. The role of crossing vessels in endopyelotomy failure is very controversial. To better understand the pathophysiology of endopyelotomy failure, the expression of transforming growth factor-beta (TBG beta) in patients with a failed endopyelotomy and crossing vessels was compared with that in patients without crossing vessels, as well as those having primary pyeloplasty or a normal ureteropelvic junction (UPJ). MATERIALS AND METHODS: The expression of TGF beta was detected immunohistochemically in slide-mounted thin sections (4 microns) cut from paraffin-blocked adult UPJ segments obtained during primary pyeloplasty (N = 11), secondary pyeloplasty after failed endopyelotomy with documented crossing vessels (N = 10), secondary pyeloplasty after failed endopyelotomy without crossing vessels (N = 11), and normal UPJs removed during nephrectomy for purposes unrelated to obstruction (N = 11). Expression was graded on a scale of 0 to 4. RESULTS: The combined failed endopyelotomy group had a significantly (P < 0.05) lower level of TGF beta (1.9 +/- 0.7) than did primary obstructed UPJs (2.6 +/- 0.7). The TGF beta level in the crossing vessels group (1.9 +/- 0.7) did not differ from that in the group without crossing vessels (1.8 +/- 0.7), nor did it differ from that in the group with normal UPJs (1.6 +/- 0.7). As expected, primary obstructed UPJs had a significantly higher level of TGF beta than normal ones (P < 0.02). CONCLUSIONS: Obstructed UPJs that had failed endopyelotomy had a similarly reduced level of TGF beta whether or not crossing vessels were present. These data suggest that an elevation of TGF beta in obstructed UPJs may be necessary for successful tissue repair after endopyelotomy and that the presence of crossing vessels is probably not relevant.
Ibuprofen combined with antibiotics suppresses renal scarring due to ascending pyelonephritis in rats.
PURPOSE: In acute pyelonephritis renal scarring may be decreased by immediate antibiotic therapy. Unfortunately in children there is often a delay in starting treatment, which increases the likelihood of renal scarring. In rodents immediate antibiotic therapy is effective for preventing renal scar formation resulting from experimentally induced pyelonephritis. However, the same treatment beginning 72 hours after infection does not prevent renal scarring in this paradigm. We examined whether delayed administration of the nonsteroidal anti-inflammatory agent ibuprofen only or combined with antibiotics suppresses renal scarring in a model of ascending pyelonephritis in rats. MATERIALS AND METHODS: An inoculum of 5x10(9) organisms per ml. of Escherichia coli strain BH-5 was instilled into the bladder of rats and the urethra was occluded for 4 hours. Groups of animals were and were not treated with 15 mg./kg. cefadroxil or 10 mg./kg. ibuprofen given twice daily for 5 days, or the 2 drugs combined. Treatment began 72 hours after inoculation. In an additional group of rats sterile phosphate buffered saline was instilled into the bladder. In each rat the kidneys were examined grossly and microscopically 6 weeks later. RESULTS: Combined antibiotics and ibuprofen significantly inhibited gross renal scarring compared with no treatment or antibiotics only (p<0.05). No difference in renal scarring was detected in animals that received no treatment versus those that received antibiotics or ibuprofen only (p>0.05). CONCLUSIONS: Renal scarring resulting from acute pyelonephritis in this rat model is not decreased by delayed treatment with antibiotics only. The addition of ibuprofen to antibiotic therapy is effective for decreasing renal scarring due to acute pyelonephritis even when treatment is delayed for 72 hours.
Necrosis of the urethra and tissue sloughing: a delayed complication after transurethral microwave thermotherapy.
Transurethral microwave thermotherapy is a minimally invasive treatment for benign prostatic hyperplasia designed to destroy hyperplastic tissue without damaging the urethra. We present an unexpected complication of prostatic urethral necrosis and tissue sloughing after thermotherapy and discuss its possible cause.
Endopyelotomy failure is associated with reduced transforming growth factor-beta.
PURPOSE: Approximately 15% of patients with ureteropelvic junction obstruction have endopyelotomy failure and require an additional surgical procedure to remove the obstruction. Transforming growth factor-beta (TGF-beta), a cytokine which stimulates mesenchymal cell proliferation and extracellular matrix deposition, increases in the renal pelvis in response to obstruction. However, TGF-beta also is implicated in smooth muscle regeneration and wound healing. To understand the pathophysiology of ureteropelvic junction obstruction and determine why endopyelotomy fails in some obstructed ureteropelvic junctions, TGF-beta expression in obstructed and normal ureteropelvic junction segments was compared. MATERIALS AND METHODS: Immunohistochemical staining using a rabbit polyclonal anti-TGF-beta was performed on deparafinfized 4 microm. sections of paraffin blocked ureteropelvic junction segments. Human obstructed ureteropelvic junction segments were removed during primary pyeloplasties (11) and secondary pyeloplasties after endopyelotomy failure (11). Normal ureteropelvic junction segments were removed during nephrectomy for purposes unrelated to obstruction (11). Grading on a scale of 0 to 4 was performed by a physician blinded to the source of the specimen. RESULTS: Mean TGF-beta expression plus or minus standard error of the mean was significantly increased (p <0.02) in obstructed ureteropelvic junctions from primary pyeloplasties (2.6+/-0.7) compared to normal ureteropelvic junctions (1.6+/-0.7), as expected. However, TGF-beta expression in the endopyelotomy failure group (1.8+/-0.6) was not significantly different from that in normal ureteropelvic junctions and was significantly lower (p <0.05) than that in obstructed ureteropelvic junctions from primary pyeloplasties. CONCLUSIONS: Obstructed ureteropelvic junctions in cases of endopyelotomy failure have decreased expression of TGF-beta compared with other obstructed ureteropelvic junctions. These data suggest that an elevation of TGF-beta in obstructed ureteropelvic junctions may be necessary for successful tissue repair after endopyelotomy.