Variable Expression of Neural Cell Adhesion Molecule Isoforms in Renal Tissue: Possible Role in Incipient Renal Fibrosis.

Rare neural cell adhesion molecule (NCAM) positive cells have been previously described within the normal human adult kidney interstitium, speculating that they could increase in the interstitium with incipient interstitial renal fibrosis (IRF). In the present study, among 93 biopsy samples of various kidney diseases, NCAM+ interstitial cells were detected in 62.4% cases. An increased number of NCAM+ cells was significantly observed only in incipient IRF compared to normal renal tissues and advanced IRF stages (p<0.001), independently of underlying diseases (p = 0.657). All three major NCAM isoforms' RT-PCR bands were visible either in normal or in kidneys with incipient IRF, albeit their mRNA expression levels measured by qRT-PCR were different. Applying qRT-PCR on pure NCAM+ cells population, obtained by laser capture microdissection, significant mRNA over-expression of NCAM140kD isoform was found in NCAM+ cells within incipient IRF (p = 0.004), while NCAM120kD and NCAM180kD isoforms were not changed significantly (p = 0.750; p = 0.704; respectively). Simultaneously, qRT-PCR also showed significant αSMA (p = 0.014) and SLUG (p = 0.004) mRNAs up-regulation within the NCAM+ cells of incipient IRF, as well as highly decreased matrix metalloproteinases (MMP) -2 and -9 mRNAs (p = 0.028; p = 0.036; respectively). However, using double immunofluorescence MMP-9 could still be detectable on the protein level in rare NCAM+ cells within the incipient IRF. Further characterization of NCAM+ cells by double immunofluorescent labeling revealed their association with molecules involved in fibrosis. Fibroblast growth factor receptor 1 (FGFR1) and α5ß1 integrin were extensively expressed on NCAM+ cells within the incipient IRF areas, whereas human epididymis protein-4 (HE4) was found to be present in few NCAM+ cells of both normal and interstitium with incipient fibrosis. Heterogeneity of NCAM+ interstitial cells in normal and incipient IRF, concerning molecules related to fibrosis and variable expression of NCAM isoforms, could suggest diverse role of NCAM+ cells in homeostasis and in regulation of renal fibrosis in diseased kidneys.

Polymorphisms of HLA-DRB1, -DQA1 and -DQB1 in inhabitants of Astana, the capital city of Kazakhstan.

BACKGROUND: Kazakhstan has been inhabited by different populations, such as the Kazakh, Kyrgyz, Uzbek and others. Here we investigate allelic and haplotypic polymorphisms of human leukocyte antigen (HLA) genes at DRB1, DQA1 and DQB1 loci in the Kazakh ethnic group, and their genetic relationship between world populations. METHODOLOGY/PRINCIPAL FINDINGS: A total of 157 unrelated Kazakh ethnic individuals from Astana were genotyped using sequence based typing (SBT-Method) for HLA-DRB1, -DQA1 and -DQB1 loci. Allele frequencies, neighbor-joining method, and multidimensional scaling analysis have been obtained for comparison with other world populations. Statistical analyses were performed using Arlequin v3.11. Applying the software PAST v. 2.17 the resulting genetic distance matrix was used for a multidimensional scaling analysis (MDS). Respectively 37, 17 and 19 alleles were observed at HLA-DRB1, -DQA1 and -DQB1 loci. The most frequent alleles were HLA-DRB1*07:01 (13.1%), HLA-DQA1*03:01 (13.1%) and HLA-DQB1*03:01 (17.6%). In the observed group of Kazakhs DRB1*07:01-DQA1*02:01-DQB1*02:01 (8.0%) was the most common three loci haplotype. DRB1*10:01-DQB1*05:01 showed the strongest linkage disequilibrium. The Kazakh population shows genetic kinship with the Kazakhs from China, Uyghurs, Mongolians, Todzhinians, Tuvinians and as well as with other Siberians and Asians. CONCLUSIONS/SIGNIFICANCE: The HLA-DRB1, -DQA1 and -DQB1 loci are highly polymorphic in the Kazakh population, and this population has the closest relationship with other Asian and Siberian populations.

Tet3-mediated hydroxymethylation of epigenetically silenced genes contributes to bone morphogenic protein 7-induced reversal of kidney fibrosis.

Methylation of CpG island promoters is an epigenetic event that can effectively silence transcription over multiple cell generations. Hypermethylation of the Rasal1 promoter contributes to activation of fibroblasts and progression of kidney fibrosis. Here, we explored whether such causative hypermethylation could be reversed through endogenous mechanisms and whether such reversal of hypermethylation is a constituent of the antifibrotic activity of bone morphogenic protein 7 (BMP7). We show that successful inhibition of experimental kidney fibrosis through administration of BMP7 associates with normalization of Rasal1 promoter hypermethylation. Furthermore, this reversal of pathologic hypermethylation was achieved specifically through Tet3-mediated hydroxymethylation. Collectively, our findings reveal a new mechanism that may be exploited to facilitate therapeutic DNA demethylation to reverse kidney fibrosis.

B cells contribute to heterogeneity of IL-17 producing cells in rheumatoid arthritis and healthy controls.

Secretion of the proinflammatory cytokine Interleukin-17A (IL-17A) is the hallmark of a unique lineage of CD4 T cells designated Th17 cells, which may play a crucial role in the pathogenesis of rheumatoid arthritis (RA) and many autoimmune diseases. Recently, IL-17-producing cells other than T cells have been described, including diverse innate immune cells. Here, we show that the cellular sources of IL-17A in RA include a significant number of non-T cells. Multicolour fluorescence analysis of IL-17-expressing peripheral blood mononuclear cells (PBMC) revealed larger proportions of IL-17(+)CD3(-) non-T cells in RA patients than in healthy controls (constitutive, 13.6% vs. 8.4%, and after stimulation with PMA/ionomycin 17.4% vs. 7.9% p < 0.001 in both cases). The source of IL-17 included CD3(-)CD56(+) NK cells, CD3(-)CD14(+) myeloid cells as well as the expected CD3(+)CD4(+) Th17 cells and surprisingly a substantial number of CD3(-)CD19(+) B cells. The presence of IL-17A-expressing B cells was confirmed by specific PCR of peripheral MACS-sorted CD19(+) B cells, as well as by the analysis of different EBV-transformed B cell lines. Here we report for the first time that in addition to Th17 cells and different innate immune cells B cells also contribute to the IL-17A found in RA patients and healthy controls.

Identification of human epididymis protein-4 as a fibroblast-derived mediator of fibrosis.

The functional contribution of myofibroblasts in fibrosis is not well understood. Using a new genetic mouse model to track and isolate myofibroblasts, we performed gene expression profiling followed by biological validation to identify HE4 (encoding human epididymis protein 4, also known as WAP 4-disulfide core domain-2 or Wfdc2) as the most upregulated gene in fibrosis-associated myofibroblasts. The HE4 gene encodes for a putative serine protease inhibitor that is upregulated in human and mouse fibrotic kidneys and is elevated in the serum of patients with kidney fibrosis. HE4 suppresses the activity of multiple proteases, including serine proteases and matrix metalloproteinases, and specifically inhibits their capacity to degrade type I collagen. In particular, we identified two serine proteases, Prss35 and Prss23, as HE4 targets with functional relevance in kidney fibrosis. Administration of HE4-neutralizing antibodies accelerated collagen I degradation and inhibited fibrosis in three different mouse models of renal disease. Collectively these studies suggest that HE4 is a potential biomarker of renal fibrosis and a new therapeutic target.

Identification and validation of six proteins as marker for endemic nephropathy.

Endemic nephropathy (EN) is defined as a slow progressive renal tubulointestitial disease that mainly occurs in the restricted areas of the Balkan Peninsula. The complexity of the pathogenesis of EN makes its earlier diagnosis very difficult. Urine samples from healthy volunteers from EN regions, EN patients with proteinuria less than 150 mg/L and EN patients with proteinuria more than 150 mg/L, patients with acute kidney injury, patients with diabetic nephropathy and healthy volunteers from Germany were collected. The urinary proteome analyses were performed using 2-D DIGE and mass spectrometry. The validation of biomarkers was investigated by two approaches (Western blot (WB) and dot blot) in successively increasing size - and partially overlapping - sample sets. Comparative and statistical analyses of the proteomics data from the different patient groups allowed the identification of six proteins (alpha-1-microglobulin, alpha-2-glycoprotein-1, beta-2-microglobulin, mannose-binding-lectin-2, protection-of-telomeres-protein-1, and superoxide-dismutase [Cu-Zn]), which were able to discriminate EN with low and high proteinuria from the other groups with high significance (p<0.05). The reliability of the identified proteins as EN marker was underlined with high statistical significance using WB analyses (sensitivity 66.7-98% and specificity 70-100%), whereas the dot blot analyses revealed a decrease in the sensitivity and specificity of these biomarkers.

Proteomics analysis identifies PARK7 as an important player for renal cell resistance and survival under oxidative stress.

Renal fibrosis is a process that is characterized by declining excretory renal function. The molecular mechanisms of fibrosis are not fully understood. Oxidative stress pathways were reported to be involved in renal tissue deterioration and fibrosis progression. In order to identify new molecular targets associated with oxidative stress and renal fibrosis, differential proteomics analysis was performed with established renal cell lines (TK173 and HK-2). The cells were treated with oxidative stress triggering factor H(2)O(2) and the proteome alterations were investigated. Two dimensional protein maps were generated and differentially expressed proteins were processed and identified using mass spectrometry analysis combined with data base search. Interestingly the increase of ROS in the renal cell lines upon H(2)O(2) treatment was accompanied by alteration of a large number of proteins, which could be classified in three categories: the first category grouped the proteins that have been described to be involved in fibrogenesis (e.g. ACTA2, VIN, VIM, DES, KRT, COL1A1, COL4A1), the second category, which was more interesting involved proteins of the oxidative stress pathway (PRDX1, PRDX2, PRDX6, SOD, PARK7, HYOU1), which were highly up-regulated under oxidative stress, and the third category represented proteins, which are involved in different other metabolic pathways. Among the oxidative stress proteins the up-regulation of PARK7 was accompanied by a shift in the pI as a result of oxidation. Knockdown of PARK7 using siRNA led to significant reduction in renal cell viability under oxidative stress. Under H(2)O(2) treatment the PARK7 knockdown cells showed up to 80% decrease in cell viability and an increase in apoptosis compared to the controls. These results highlight for the first time the important role of PARK7 in oxidative stress resistance in renal cells.

Methylation determines fibroblast activation and fibrogenesis in the kidney.

Fibrogenesis is a pathological wound repair process that fails to cease, even when the initial insult has been removed. Fibroblasts are principal mediators of fibrosis, and fibroblasts from fibrotic tissues fail to return to their quiescent stage, including when cultured in vitro. In a search for underlying molecular mechanisms, we hypothesized that this perpetuation of fibrogenesis is caused by epigenetic modifications. We demonstrate here that hypermethylation of RASAL1, encoding an inhibitor of the Ras oncoprotein, is associated with the perpetuation of fibroblast activation and fibrogenesis in the kidney. RASAL1 hypermethylation is mediated by the methyltransferase Dnmt1 in renal fibrogenesis, and kidney fibrosis is ameliorated in Dnmt1(+/-) heterozygous mice. These studies demonstrate that epigenetic modifications may provide a molecular basis for perpetuated fibroblast activation and fibrogenesis in the kidney.

The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase.

We have recently identified 2 distinct CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-) MSC subsets in primary femur-derived bone marrow (BM), which differ in their expression pattern and morphology as well as in their clonogenic and differentiation capacity. Here, we show that MSCA-1 is identical to tissue non-specific alkaline phosphatase (TNAP), an ectoenzyme known to be expressed at high levels in liver, bone, and kidney as well as in embryonic stem (ES) cells. SDS-PAGE of WERI-RB-1 cell lysate and supernatant from phosphatidylinositol-specific phospholipase C (PI-PLC)-treated WERI-RB-1 cells resulted in the appearance of a prominent 68-kDa band. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) sequence analysis revealed TNAP-specific peptides. Screening of the MSCA-1-specific antibody W8B2 on HEK-293 cells transfected with the full-length coding sequence of TNAP showed specific reactivity with transfected but not with parent cell line. In addition, TNAP-specific mRNA expression was selectively detected in the transfectant line. In agreement with these findings, enzymatic activity of TNAP was exclusively detected in sorted MSCA-1(+) BM cells but not in the MSCA-1(-) negative fraction. Surface marker analysis revealed coexpression of the embryonic marker SSEA-3 but not SSEA-4, TRA-1-60, and TRA-1-81. In endometrium, TNAP is expressed at intermediate levels on CD146(+) cells and at high levels in the luminal space of glandular epithelia. Our results demonstrate that TNAP is a selective marker for the prospective isolation of BM-derived MSC and MSC-like cells in endometrium.

The integrin alpha9beta1 on hematopoietic stem and progenitor cells: involvement in cell adhesion, proliferation and differentiation.

BACKGROUND: Hematopoietic stem and progenitor cells can interact with their microenvironment via integrins which are adhesion receptors consisting of alpha and beta subunits. Current knowledge suggests that the integrin subunits alpha4 and alpha6 expressed on hematopoietic stem and progenitor cells have distinct roles in retaining stem cells in the bone marrow. The aim of our study was to gain insight into the expression and functions of the integrin subunits alpha7-alpha11 within the endosteal stem cell niche. DESIGN AND METHODS: Human osteoblasts isolated from trabecular bone and hematopoietic stem and progenitor cells purified from umbilical cord blood or bone marrow aspirates were analyzed for the expression of integrin alpha7-alpha11 chains by reverse transcriptase polymerase chain reaction. The involvement of the integrin alpha9beta1 in hematopoietic stem and progenitor cell adhesion, proliferation and differentiation was analyzed in functional assays. RESULTS: Transcripts for all investigated integrin chains were found in primary osteoblasts. Highly purified hematopoietic stem and progenitor cells, however, expressed only transcripts encoding integrin subunits alpha7 and alpha9. Flow cytometric analysis verified extracellular expression of the integrin alpha9beta1 on hematopoietic stem and progenitor cells. Cell-cell adhesion assays with osteoblasts and dye-labeled CD34(+) hematopoietic stem and progenitor cells in the presence of function-blocking antibodies revealed a role of integrin alpha9 in hematopoietic stem and progenitor cell adhesion to osteoblasts. Furthermore, the addition of anti-integrin alpha9 antibodies significantly inhibited proliferation and in vitro differentiation of CD34(+) hematopoietic stem and progenitor cells. CONCLUSIONS: The integrin alpha9beta1 has been identified as a new member of the integrin beta1-subfamily expressed on human hematopoietic stem and progenitor cells. The functional studies strongly suggest that integrin alpha9beta1 contributes to adhesion and differentiation of hematopoietic stem and progenitor cells in the endosteal stem cell niche.

Low-affinity peptides and T-cell selection.

The dual requirement for T cells to recognize a particular major histocompatibility complex (MHC) antigen presenting a foreign peptide and to lack strong reactivity with a complex of the same molecule when bound to a self-peptide, is attained by thymic positive and negative selection processes, the molecular details of which are currently only partially understood. However, the discovery of the thymoproteasome and our improved understanding of the dynamics of peptide presentation permit us to suggest that the biophysical properties of the MHC:peptide class I complexes engaged in positive T-cell selection will be distinct from those involved in negative selection, hence imposing differential barriers for T cells.

HLA ligand profiles of primary renal cell carcinoma maintained in metastases.

In recent years, several approaches have been taken in the peptide-based immunotherapy of metastatic renal cell carcinoma (RCC), although little is known about HLA presentation on metastases compared to primary tumor and normal tissue of RCC. In this study we compared primary tumor, normal tissue and metastases with the aim of identifying similarities and differences between these tissues. We performed this comparison for two RCC patients on the level of the HLA ligandome using mass spectrometry and for three patients on the level of the transcriptome using oligonucleotide microarrays. The quantitative results show that primary tumor is more similar to metastasis than to normal tissue, both on the level of HLA ligand presentation and mRNA. We were able to characterize a total of 142 peptides in the qualitative analysis of HLA-presented peptides. Six of them were significantly overpresented on metastasis, among them a peptide derived from CD151; fourteen were overpresented on both primary tumor and metastasis compared to normal tissue, among them an HLA ligand derived from tumor protein p53. Thus, we could demonstrate that peptide-based immunotherapy might affect tumor as well as metastasis of RCC, but not healthy kidney tissue. Furthermore we were able to identify several peptides derived from tumor-associated antigens that are suitable for vaccination of metastatic RCC.

Primary CNS lymphoma and HLA class I and II alleles in a German cohort of immunocompetent patients.

Human leukocyte antigens (HLA) are involved in the regulation of immune response to infection and in malignant transformation. Several HLA alleles are associated with immunological or malignant diseases. The aim of the present study was to evaluate a potential association of HLA class I and II alleles with primary central nervous system lymphoma (PCNSL) in immunocompetent patients. We therefore analyzed particular HLA-A, HLA-B and HLA-DRB1 alleles in 82 PCNSL patients and compared the data to those in 327 population controls. No significant difference between these two groups was found using Pearson's chi(2) test. These data do not support the hypothesis that HLA alleles play a major role in the pathogenesis of PCNSL.

Proteomic analysis to improve adequacy of hemo- and peritoneal dialysis: Removal of small and high molecular weight proteins with high- and low-flux filters or a peritoneal membrane.

The reduction of morbidity and mortality in patients undergoing hemo- or peritoneal dialysis is strongly related to an efficient and selective clearance of uremic toxins. We used proteomics methods to analyze and further characterize the dialytic removal of still undefined middle and high molecular weigh proteins as a basis for further improvement of dialysis assessment. Dialysates from 26 hemodialysis patients treated with different types of low- (F6HPS®) and high-flux (FX80®, APS650®, FX60®) filters as well as peritoneal fluids from 10 continuous ambulatory peritoneal dialysis (CAPD) patients were analyzed by SELDI-TOF and 2-DE. The protein patterns showed selective differences in the proteins cleared depending on the dialysis method used and the filter membrane. While SELDI analyses of dialysates from the F6HPS revealed almost no protein clearance, high-flux filters and CAPD dialysates showed protein release of different molecular weight ranges. Furthermore, 2-DE and MS analysis identified 48 different proteins from the dialysate of high-flux filters and 21 from peritoneal dialysis fluids. In F6HPS dialysates, however, only few proteins could be identified.

Cadherin-9 is a novel cell surface marker for the heterogeneous pool of renal fibroblasts.

BACKGROUND: Interstitial fibroblasts are a minor, but nevertheless very important, component of the kidney. They secrete and remodel extracellular matrix and they produce active compounds such as erythropoietin. However, studying human renal fibroblasts has been hampered by the lack of appropriate surface markers. METHODS AND FINDINGS: The expression of cadherin-9 in various human renal cell lines and tissues was studied on the mRNA level by RT-PCR and on the protein level with the help of newly generated cadherin-9 antibodies. The classical type II cadherin-9, so far only described in the neural system, was identified as a reliable surface marker for renal fibroblasts. Compared to FSP1, a widely-used cytosolic renal fibroblast marker, cadherin-9 showed a more restricted expression pattern in human kidney. Under pathological conditions, cadherin-9 was expressed in the stroma of renal cell carcinoma, but not in the tumor cells themselves, and in renal fibrosis the percentage of cadherin-9-positive cells was clearly elevated 3 to 5 times compared to healthy kidney tissue. Induction of epithelial mesenchymal transition in renal epithelial cells with cyclosporin-A, which causes renal fibrosis as a side effect, induced cadherin-9 expression. Functional studies following siRNA-mediated knockdown of cadherin-9 revealed that it acts in the kidney like a typical classical cadherin. It was found to be associated with catenins and to mediate homophilic but not heterophilic cell interactions. CONCLUSIONS: Cadherin-9 represents a novel and reliable cell surface marker for fibroblasts in healthy and diseased kidneys. Together with the established marker molecules FSP1, CD45 and alpha smooth muscle actin, cadherin-9 can now be used to differentiate the heterogenic pool of renal fibroblasts into resident and activated fibroblasts, immigrated bone marrow derived fibroblast precursors and cells in different stages of epithelial mesenchymal transition.

Neural cell adhesion molecule expression on renal interstitial cells.

BACKGROUND: At early stages of kidney development, the neural cell adhesion molecule (NCAM) is highly expressed on cells of the metanephrogenic mesenchyme. During maturation of the fetal kidney, NCAM gradually disappears. So far, it has been widely accepted that NCAM in the adult kidney is only expressed by nerves, and not by other cell types. METHODS: NCAM expression was analysed in human adult healthy and diseased kidneys by immunohistochemistry and western blot analysis. NCAM+ renal interstitial cells were further characterized by double immunofluorescent staining using antibodies against neurofilaments, alpha smooth muscle actin, vimentin, alpha5beta1 integrin, CD68, CD11c, HLA-DR and the potential progenitor cell markers CD34, CD117, CD133, CD24, nestin and cadherin-11. RESULTS: In adult human kidneys, NCAM expression is restricted to rare interstitial cells with dendritic morphology, which are neurofilament-negative and predominantly localized on the corticomedullary junction. They are also negative for fibroblast cell markers, but co-express the haematopoietic stem cell markers CD34 and CD133. The number of NCAM+ interstitial cells increased in the initial phases of interstitial fibrosis. Western blot analysis of renal tissues with incipient interstitial fibrosis tissues showed the expression of the 140 kDa NCAM isoform. CONCLUSIONS: These data indicate that a rare subpopulation of NCAM+ interstitial cells could represent renal progenitors, and that NCAM+ interstitial cells can participate in the initial phase of interstitial fibrosis.

[Expression of Fas and Fas-L in renal cell carcinoma].

INTRODUCTION: The previous investigations revealed that Fas-L expression on tumor cells can be one of the reasons of tumor growth, or tumor regression, with or without activation of the immune response. OBJECTIVE: The objective of our study was to investigate the expression of Fas and Fas-L in situ in normal human renal tissue as well as in different types of renal cell carcinoma (RCC) according to tumor grading. METHOD: Expression of Fas and Fas-L was examined in 25 RCCs classified according to nuclear grades: G1-G3 and to cell type: 17 clear cells, 3 chromophilics (2 eosinophilics, 1 basophilic), 2 chromophobes and 3 spindle cells. Ten normal human kidneys were analyzed, too. Indirect immunoperoxidase technique was applied. Spread and intensity of staining of Fas and Fas-L molecules expression were scored semiquantitatively. RESULTS: Distribution of Fas expression in these RCC was typically diffuse. However, Fas-L was almost completely absent in clear cell RCC. In 3 clear cell RCC, some tumor stromal cells exhibited strong expression of Fas-L. On the contrary, chromophilic, chromophobe and spindle cell RCCs grading from G2-G3, manifested variable combinations of Fas and Fas-L expression. CONCLUSION: The most of clear cell type low grade RCCs manifested intensive and extensive expression of Fas and completely absence of Fas-L. However, RCCs of high grade malignancy belonging to the clear cell, eosinophilic, chromophobe or spindle cell types can have various combinations of Fas and Fas-L expression. It may probably lead to development of different mechanisms of avoidance of immune response to RCC.

Characterization of a conduit system containing laminin-5 in the human thymus: a potential transport system for small molecules.

T cells develop in the thymus in a highly specialized cellular and extracellular microenvironment. The basement membrane molecule, laminin-5 (LN-5), is predominantly found in the medulla of the human thymic lobules. Using high-resolution light microscopy, we show here that LN-5 is localized in a bi-membranous conduit-like structure, together with other typical basement membrane components including collagen type IV, nidogen and perlecan. Other interstitial matrix components, such as fibrillin-1 or -2, tenascin-C or fibrillar collagen types, were also associated with these structures. Three-dimensional (3D) confocal microscopy suggested a tubular structure, whereas immunoelectron and transmission electron microscopy showed that the core of these tubes contained fibrillar collagens enwrapped by the LN-5-containing membrane. These medullary conduits are surrounded by thymic epithelial cells, which in vitro were found to bind LN-5, but also fibrillin and tenascin-C. Dendritic cells were also detected in close vicinity to the conduits. Both of these stromal cell types express major histocompatibility complex (MHC) class II molecules capable of antigen presentation. The conduits are connected to blood vessels but, with an average diameter of 2 mum, they are too small to transport cells. However, evidence is provided that smaller molecules such as a 10 kDa dextran, but not large molecules (>500 kDa), can be transported in the conduits. These results clearly demonstrate that a conduit system, which is also known from secondary lymphatic organs such as lymph nodes and spleen, is present in the medulla of the human thymus, and that it might serve to transport small blood-borne molecules or chemokines to defined locations within the medulla.