Microanatomic Location Research Articles

Validation of the S Classification of Sentinel Lymph Node and Microanatomic Location of Sentinel Lymph Node Metastases to Predict Additional Lymph Node Involvement and Overall Survival in Breast Cancer Patients

Most patients with a positive sentinel lymph node (SN) have no further metastases in the axillary lymph nodes and may therefore not benefit from axillary lymph node dissection. In patients with melanoma, evaluation of the centripetal depth of tumor invasion in the SN, also known as the S classification of SN, and microanatomic localization of SN metastases were shown to predict non-SN involvement. This phenomenon has been less extensively studied in breast cancer. We sought to validate the S classification and microanatomic location of SN metastases in breast cancer patients with regard to their predictive value for non-SN involvement and overall survival (OS). A total of 236 patients with positive SN followed by axillary lymph node dissection were reevaluated according to the S classification and the microanatomic location of SN (subcapsular, parenchymal, combined subcapsular and parenchymal, multifocal, extensive) metastases to predict the likelihood of non-SN metastases and OS. S classification and the microanatomic location of SN metastases were significantly correlated with non-SN status (P<0.001). Especially patients with a maximum depth of invasion ≤0.3mm (stage I according to the S classification) and those with SN metastases only in subcapsular location had a low probability of further non-SN metastases (7.8 and 6.1%) and a good prognosis for OS. S classification and microanatomic location of SN metastases predicts the likelihood of non-SN involvement. Especially patients with subcapsular or S stage I metastases have a low probability of non-SN metastases and a good prognosis for OS.

Factors predicting non-sentinel lymph node involvement in sentinel node positive breast carcinoma

In routine practice, axillary lymph node dissection is performed in early invasive breast cancer patients with positive sentinel node biopsy. However, sentinel node is the only involved axillary node in 40-70% of patients, and determining factors that predict axillary non-sentinel node involvement will therefore prevent unnecessary axillary lymph node dissection and decrease morbidity. In this study, 119 invasive breast cancer patients with sentinel node metastasis who underwent axillary lymph node dissection between 1998-2009 at our institution were studied. Primary tumor characteristics and features of the metastatic tumors in sentinel nodes, such as microanatomic location, size of metastasis, and the ratio of metastatic tumor area to the total sentinel node area were evaluated. Student's t-test and multivariate logistic regression were used for statistical analysis. The mean age of the patients was 50.7 years (28-80). Forty-three patients (36%) had invasive ductal and 25 patients (21%) had invasive lobular carcinoma. Most of the patients had either pT1 (44%) or pT2 (54%) tumors. Fifty-four patients (45%) had no further positive nodes in the axilla. The metastatic deposits in the sentinel node were subcapsular in 16 patients (13%). The percent area of sentinel node occupied by tumor (p < 0.001), number of sentinel nodes (p=0.041), and microanatomic location of metastatic tumor (p=0.002) were significantly associated with non-sentinel node metastasis in univariate analysis. The percent area of sentinel node occupied by tumor (p < 0.001) and number of sentinel nodes (p=0.033) remained significantly associated with non-sentinel node involvement in multivariate analysis. In patients with invasive breast cancer and positive sentinel node, area percent of sentinel node occupied by tumor, and the number of sentinel nodes removed are independently predictive of non-sentinel node involvement.

Multimarker Reverse Transcriptase-Polymerase Chain Reaction Assay in Lymphatic Drainage and Sentinel Node Tumor Burden

PurposeWe assessed molecular (presence of melanoma cells markers in lymph fluid [LY]) and pathological features (sentinel lymph node [SN] tumor burden according to Rotterdam criteria, metastases microanatomic location) and correlated them with survival and melanoma prognostic factors in a group of patients with positive SN biopsy.MethodsWe analyzed 368 consecutive SN-positive patients after completion lymph node dissection (CLND). In 321 patients we obtained data on SLN microanatomic location/tumor burden (only 7 cases had metastases <0.1 mm); in 137 we additionally analyzed 24-hour collected LY after CLND (multimarker reverse transcriptase-polymerase chain reaction [MM-RT-PCR] with primers for tyrosinase, MART1 (MelanA), and uMAGE mRNA (27.7% positive samples)]. Median follow-up time was 41 months.ResultsAccording to univariate analysis, the following factors had a negative impact on overall survival (OS): higher Breslow thickness (P = .0001), ulceration (P < .0001), higher Clark level (P = .008), male gender (P = .0001), metastatic lymph nodes >1 (P < .0001), nodal metastases extracapsular extension (P < .0001), metastases to additional non-SNs (P = .0004), micrometastases size ≥0.1 mm (P = .0006), and positive LY MM-RT-PCR (P = .0007). SN tumor burden showed linear correlation with increasing Breslow thickness (P = .01). The 5-year OS rates for SLN tumor burden <0.1 mm, 1–1.0 mm, and >1.0 mm were 84%/66%/44%, respectively, and for positive and negative LY MM-RT-PCR 47%/0%, respectively. The independent factors for shorter OS (multivariate analysis): male gender, primary tumor ulceration, number of involved nodes ≥4, micrometastases size >1.0 mm, and, in additional model including molecular analysis—positive MM-RT-PCR results (hazard ratio [HR] 3.2), micrometastases size >1.0 mm (HR 1.13), and primary tumor ulceration (HR 2.17). Similar results were demonstrated for disease-free survival (DFS) data.ConclusionsSN tumor burden categories according to Rotterdam criteria and the positive result of LY MM-RT-PCR assay demonstrated additional, independent prognostic value in SN-positive melanoma patients, showing significant correlation with shorter DFS and OS.

TRANSVERSE ISOTROPIC ELASTIC PROPERTIES OF VERTEBRAL TRABECULAR BONE MATRIX MEASURED USING MICROINDENTATION UNDER DRY CONDITIONS (EFFECTS OF AGE, GENDER, AND VERTEBRAL LEVEL)

The mechanical properties of bone extracellular matrix have become of increasing interest for the understanding of vertebral fracture risk. Depth-sensing indentation techniques allow the measurement of directional elastic properties of trabecular bone ex vivo with a high spatial resolution. Transverse isotropic elastic properties of vertebral trabecular bone obtained from two orthogonal directions were investigated using microindentation under dry conditions focusing on the influence of microanatomical location, age, gender, vertebral level, and anatomic direction on these properties. Biopsies were obtained from 104 human vertebrae (T1–L3) with a median age of 65 (21–94) years. Significantly, higher indentation moduli were found for indentations on axial than on transverse cross-sections of trabeculae (p &lt; 0.01). Indentation moduli in the core were 1.05 to 1.12 times higher than in the periphery (p &lt; 0.01). No difference in stiffness could be detected between males and females (p &gt; 0.05) and different ages (p &gt; 0.5). Vertebral level showed a weak correlation (p = 0.073, r2 ≈ 0.17). These results provide insights in the transverse isotropic properties of trabecular bone matrix related to age, gender, microanatomical location, and anatomic direction for a broad spectrum of human vertebrae.

Pulmonary Cyclooxygenase-1 (COX-1) and COX-2 Cellular Expression and Distribution After Respiratory Syncytial Virus and Parainfluenza Virus Infection

Prostaglandins (PGs) play an important role in pulmonary physiology and various pathophysiological processes following infection. The initial step in the biosynthesis of PGs is regulated by two distinct cyclooxygenase enzymes, cyclooxygenase-1 (COX-1) and COX-2. The goal of this study was to investigate the pulmonary cellular localization and distribution of COX-1 and COX-2 in a neonatal lamb model following respiratory syncytial virus (RSV) and parainfluenza virus 3 (PI3) infection, organisms that also cause significant respiratory disease in children. No significant differences were seen in pulmonary COX-1 expression at various microanatomical locations following RSV or PI3 infection compared to controls. In contrast, COX-2 was upregulated following RSV and PI3 infection. Strong expression was restricted to bronchial and bronchiolar epithelial cells and macrophages, while minimal expression was present in the same microanatomical locations in the uninfected lungs. Other microanatomical locations in both the controls and the infected lungs lacked expression. This work suggests that during RSV or PI3 infection: (1) COX-1 cellular expression is not altered, (2) COX-2 cellular expression is upregulated in airway bronchiolar and bronchial epithelial cells and macrophages, (3) respiratory epithelium along with macrophages are important microanatomical compartments regulating the host inflammatory response during viral infection, and (4) COX-2 may be a potential target for RSV and PI3 therapy.

Isolated Tumor Cells in Axillary Lymph Nodes of Breast Cancer Patients: Differential Prognostic Impact of Single Tumor Cell(s) Versus Tumor Cell Clusters, and Microanatomic Location. New Results from the Dutch MIRROR Study.

Abstract BackgroundThe MIRROR study is the largest breast cancer cohort study on pN0(i+) and pN1mi in the sentinel node era, with central pathology review plus restaging according to the 6th AJCC classification, and separate analyses on the impact of systemic adjuvant therapy. In patients not receiving systemic therapy, pN0(i+) as final N-status was shown to be an equally independent prognosticator for disease-free survival as pN1mi compared with pN0 (SABCS 2008, #23, oral). However, pN0(i+) is a heterogeneous group, consisting of single tumor cell(s) and clusters of cells, and consisting of locations in either sinus or parenchyma. We determined the differential impact of size and microanatomic location of nodal involvement on disease-free survival.MethodsWomen operated for breast cancer in all Dutch hospitals in the years 1998-2005, with favorable characteristics (tumor size 1-3 cm and differentiation grade I-II OR tumor size &amp;lt;=1 cm irrespective of grade), having undergone a sentinel node procedure were selected. For this present analysis, only patients with pN0(i+) as final nodal status who had not received systemic adjuvant therapy were included (n=513). Presence of single tumor cell(s) versus tumor cell clusters, and the microanatomic location (sinus versus parenchyma) were recorded. In addition, metastatic size was measured. The primary endpoint was 5-year disease-free survival.ResultsThe 5-year disease-free survival of patients with lymph nodes containing single tumor cell(s) (n=93) was 78.6%, as compared to a 5-year disease-free survival of 77.1% of patients with small clusters of cells (n=404). After adjustment for age, log tumor size, grade, axillary treatment, and hormone receptor status, the adjusted hazard ratio for events was 1.10 (95% CI 0.67-1.78, NS) for the presence of tumor cell clusters as compared to the presence of single tumor cell(s). When including log metastasis size within the pN0(i+) group as a continuous variable in the Cox regression model, increasing metastatic size was borderline significantly associated with reduced 5-years disease-free survival with an adjusted hazard ratio of 1.27 (95% CI 1.00 – 1.61, p=0.05). The adjusted hazard ratio for events was 0.93 (95% CI 0.57-1.51, NS) for the parenchymal (n=112) versus sinusoidal microanatomic location (n=395).ConclusionSingle tumor cell(s) bear similar prognostic information as small tumor cell clusters, even though results do suggest that within the pN0(i+) group increasing size of nodal involvement is associated with reduced survival. Microanatomic location does not seem to have prognostic relevance.Support: The Netherlands organization for health research and development (ZonMw) and the Dutch Breast Cancer Trialists' Group (BOOG) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 306.

Rehydration of vertebral trabecular bone: Influences on its anisotropy, its stiffness and the indentation work with a view to age, gender and vertebral level

For understanding the fracture risk of vertebral bodies the macroscopic mechanical properties of the cancellous core are of major interest. Due to the hierarchical nature of bone, these depend in turn on the micromechanical properties of bone extracellular matrix which is at least linear elastic transverse isotropic. The experimental determination of local elastic properties of bone ex vivo necessitates a high spatial resolution which can be provided by depth-sensing indentation techniques. Using microindentation, this study investigated the effects of rehydration on the transverse isotropic elastic properties of vertebral trabecular bone matrix obtained from two orthogonal directions with a view to microanatomical location, age, gender, vertebral level and anatomic direction in a conjoint statistics.Biopsies were gained from 104 human vertebrae (T1–L3) with a median age of 65 years (21–94). Wet elastic moduli were 29% lower (p<0.05) than dry elastic moduli. For wet indentation the ratio of mean elastic moduli tested in axial to those tested in transverse indentation direction were 1.13 to 1.23 times higher than for dry indentation. The ratio of elastic moduli tested in the core to those tested in the periphery of trabeculae was 1.05 to 1.16 times higher when testing wet. Age and gender did not show any influence on the elastic moduli for wet and dry measurements. The correlation between vertebral level and elastic moduli became weaker after rehydration (pwet<0.09, rwet2=0.14) and (pdry<0.01, rwet2=0.38). Elastic and dissipated energies were similarly affected by rehydration compared to the elastic modulus. No significant difference in the energies could be found for gender (p>0.05). Significant differences in the energies were found for age (p<0.05) after rehydration.Qualitative and quantitative insights into the transverse isotropic elastic properties of trabecular bone matrix under two testing conditions over a broad spectrum of vertebrae could be given. This study could help to further improve understanding of the mechanical properties of vertebral trabecular bone.

Expert opinion in melanoma: The sentinel node; EORTC Melanoma Group recommendations on practical methodology of the measurement of the microanatomic location of metastases and metastatic tumour burden

The sentinel node (SN) status has been recognised to be the most important prognostic factor in melanoma. Many studies have investigated additional factors to further predict survival/lymph node involvement. The EORTC Melanoma Group (MG) has formulated the following question: How should we report the microanatomic location and SN tumour burden? The EORTC MG recommends the following: the EORTC MG SN pathology protocol or a similarly extensive protocol, which has also been proven to be accurate, should be used. Only measure what you can see not what you presume. Cumulative measurements decrease the accuracy and reproducibility of measuring. The most reproducible measure is a single measurement of the maximum diameter of the largest lesion in any direction (1-D). If there is any infiltration into the parenchyma, this lesion can no longer be considered solely subcapsular. Reporting of the microanatomic location of metastases should be an assessment of the entire sentinel node, not only of the largest lesion. Multifocality reflects a scattered metastatic pattern, not to be confused with multiple cohesive foci, which fall under the regular location system. A subcapsular metastasis should have a smooth usually curved outline, not ragged or irregular. We recommend all pathologists to report the following items per positive SN for melanoma patients: the microanatomic location of the metastases according to Dewar et al. for the entire node, the SN Tumour Burden according to the Rotterdam Criteria for the maximum diameter of the largest metastasis expressed as an absolute number, and the SN Tumour Burden stratified per category; <0.1 mm or 0.1–1.0 mm or >1.0 mm.

Long-term follow-up of patients with minimal sentinel node tumor burden (&lt; 0.1mm) according to Rotterdam criteria: A study of the EORTC Melanoma Group

9005 Background: Many studies have identified Sentinel Node (SN) tumor burden as a prognostic factor for additional non- SN (NSN) positivity and / or disease-free (DFS) and melanoma specific survival (MSS). It remains unclear if pts with minimal SN tumor burden can safely be managed without Completion Lymph Node Dissection (CLND). Pts with minimal SN tumor burden might be at risk for late recurrences (&gt; 5 years). Methods: Slides of 595 SN positive patients were reviewed for this pan-European study collaboration in 5 major centers. Slides were reviewed for the microanatomic location and SN tumor burden according to the Rotterdam Criteria (&lt; 0.1mm, 0.1 - 1.0mm and &gt; 1.0 mm) for the maximum diameter of the largest metastasis. MSS, DFS and distant metastasis-free survival (DMFS) rates were calculated, as was NSN positivity. Results: In 595 SN positive pts, the mean and median Breslow thickness was 4.73 and 3.5 mm. Ulceration was present in 51% of melanomas. 67 pts had metastases &lt; 0.1 mm (11%), 226 pts (38%) had 0.1 - 1.0 mm metastases and 302 pts had metastases &gt; 1.0 mm (51%). Mean and median follow-up was 48 and 40 months for all patients (range 1 - 172). Patients with metastases &lt; 0.1 mm had mean and median follow-up of 61 and 57 months, 46% (31pts) had follow up &gt; 5 years and 25% (17 pts) had follow-up longer than 80 months (range 3 - 132). 5-year MSS rates were 94% for metastases &lt; 0.1 mm, 70% for 0.1 - 1.0 mm and 57% for &gt; 1.0 mm (p&lt;0.001). 5-year DMFS rate was 91% for metastases &lt; 0.1 mm. NSN positivity occurred in 5% of &lt; 0.1mm, 17% of 0.1 - 1.0 and 29% of metastases &gt; 1.0 mm (p&lt;0.001). Conclusions: This large multicenter experience (n=595) has demonstrated that long-term follow-up of melanoma patients with minimal SN tumor burden (&lt; 0.1 mm) indicates very low relapse rates and excellent MSS, seemingly identical to SN negative patients. With prolonged follow-up, an increase in the occurrence of relapses of any kind between 5 and 10 years follow up has not been identified, and excellent 10-year survival rates are expected. No significant financial relationships to disclose.

Stage migration after minor changes in histologic estimation of tumor burden in sentinel lymph nodes

Melanoma metastasis size estimates are of prognostic significance for groups of patients, but to the authors' knowledge, measurement consensus does not exist. Maximum metastasis diameter, maximum centripetal tumor depth, microanatomic location of metastases, and complete metastasis volume were measured in 156 positive sentinel lymph nodes (SLNs) from 99 melanoma patients. The number of SLN-positive patients was increased by up to 41% using complete step-sectioning compared with less extensive protocols. Assessing maximum metastasis diameters, up to 27% of patients positive by the less extensive protocols went from 1 metastasis diameter group to a larger one when complete step-sectioning was performed. No patients were down-staged. Apparently minor protocol changes (eg, adding an extra step) led to substantial changes in maximum metastasis diameter. Similar protocol-dependent results were noted measuring the maximum centripetal tumor depth and the microanatomical location of metastases. By using semiquantitative tumor burden estimates, stage migration was always unidirectional (ie, moving from a lower to higher stage). Stereologic tumor burden estimates in step-sectioned SLNs also varied according to the number of step sections assessed, but could increase, decrease, or remain constant, so that stage migration was multidirectional. Adding extra steps to pathology protocols when assessing semiquantitative parameters leads to unidirectional stage migration ("the protocol trap"). This systematical bias makes it difficult to base treatment decisions on semiquantitative metastasis size estimates. Although based on metastatic melanoma, the principles described herein will apply when measuring nodal tumor burden in other metastasizing cancers, including breast carcinoma.

CELLULAR EXPRESSION OF RENAL, CARDIAC AND PULMONARY INDUCIBLE NITRIC OXIDE SYNTHASE IN DOUBLE‐TRANSGENIC MICE EXPRESSING HUMAN RENIN AND ANGIOTENSINOGEN GENES

1. Hypertensive mice expressing the human renin (REN) and angiotensinogen (AGT) genes are used as a model for human hypertension. 2. The aim of the present study was to investigate the cellular expression and distribution of inducible nitric oxide synthase (iNOS) using immunohistochemistry in lung, heart and kidney tissues from a model of human hypertension using male and female double-transgenic (h-Ang 204/1h-Ren6) mice and wild-type C57/BI6J mice as controls. 3. In the kidney, the pattern of iNOS expression in various renal microanatomical regions during hypertension was similar to that of age-matched controls, except in the medullary ascending limb (MAL). In hypertension, iNOS expression was downregulated in the MAL. No significant differences in iNOS expression were seen between control or hypertensive mice in various cardiac microanatomical locations. In the lungs of hypertensive mice, iNOS expression was upregulated in bronchial airway epithelium and bronchial and vascular smooth muscle cells, but downregulated in alveolar macrophages, alveolar septa and pulmonary vascular endothelial cells. Expression of iNOS was similar between male and female mice in the kidney, heart and lungs. 4. In conclusion, iNOS regulation in hypertension is complex and depends on the cell type in which it is expressed and the localization of the cell type in the cardiorenal and pulmonary systems.

The detection and significance of melanoma micrometastases in sentinel nodes

Sentinel node (SN) biopsy in melanoma patients is an accurate and minimally invasive method of staging and determining prognosis in patients with early-stage disease, and of identifying those patients who may benefit from complete regional lymph node dissection. Careful identification, removal and pathological assessment of SNs is critical to the accuracy of the technique and deficiencies in any of these steps may result in a false-negative biopsy. Pathologists should examine multiple sections from each SN, stained routinely with haematoxylin-eosin and immunohistochemically for melanoma-associated antigens. However, the most appropriate staining and sectioning protocol is not clear from the available evidence. While it appears that more extensive sectioning protocols detect more melanoma, failure rates and false-negative rates of the procedure do not appear to be significantly worse than with less extensive sampling protocols. Micromorphometrical parameters of melanoma deposits in SNs (such as their size, maximal tumour penetrative depth, microanatomical location and percentage nodal cross-sectional area) have been shown to be predictive of regional non-SN involvement and of clinical outcome. Classifying and measuring these parameters can be difficult, depending on the nature and distribution of the tumour deposits in the SN. Because the positive SNs have been removed in all patients included in studies assessing the clinical significance of tiny SN melanoma micrometastases, the likely therapeutic effect of the SN biopsy itself and/or any complete lymph node dissection, as well as lead time bias, are important confounding factors that must be considered when interpreting these studies. Limited data from some retrospective studies with relatively short follow-up suggest that some melanoma metastases may not be clinically relevant, but other studies imply a likely clinical significance of even very small SN metastases. Until there is unequivocal evidence from prospective randomised clinical trials with long term follow-up for the prognostic significance (or lack thereof) of very small SN tumour deposits, it is probably prudent to treat patients with such deposits as SN-positive.

CCR2 Mediates Conventional Dendritic Cell Recruitment and the Formation of Bronchovascular Mononuclear Cell Infiltrates in the Lungs of Mice Infected withCryptococcus neoformans

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. CCR2-deficient mice infected with C. neoformans develop a non-protective T2 immune response and persistent infection. The mechanisms responsible for this aberrant response are unknown. The objective of this study was to define the number, phenotype, and microanatomic location of dendritic cells (DC) residing within the lung of CCR2+/+ or CCR2-/- mice throughout a time course following infection with C. neoformans. Results demonstrate the CCR2-mediated recruitment of conventional DC expressing modest amounts of costimulatory molecules. DC recruitment was preceded by the up-regulation in the lung of the CCR2 ligands CCL2 and CCL7. Colocalization of numerous DC and CD4+ T cells within bronchovascular infiltrates coincided with increased expression of IL-12 and IFN-gamma. By contrast, in the absence of CCR2, DC recruitment was markedly impaired, bronchovascular infiltrates were diminished, and mice developed features of T2 responses, including bronchovascular collagen deposition and IL-4 production. Our results demonstrate that CCR2 is required for the recruitment of large numbers of conventional DC to bronchovascular infiltrates in mice mounting a T1 immune response against a fungal pathogen. These findings shed new insight into the mechanism(s) by which DC recruitment alters T cell polarization in response to an infectious challenge within the lung.

The Microanatomic Location of Metastatic Breast Cancer in Sentinel Lymph Nodes Predicts Nonsentinel Lymph Node Involvement

BackgroundThe majority of sentinel node (SN) positive breast cancer patients do not have additional non-SN involvement and may not benefit from axillary lymph node dissection (ALND). Previous studies in melanoma have suggested that microanatomic localization of SN metastases may predict non-SN involvement. The present study was designed to assess whether these criteria might also be used to be more restrictive in selecting breast cancer patients who would benefit from an ALND.MethodsA consecutive series of 357 patients with invasive breast cancer and a tumor-positive axillary SN, followed by an ALND, was reviewed. Microanatomic SN tumor features (subcapsular, combined subcapsular and parenchymal, parenchymal, extensive localization, multifocality, and the penetrative depth from the SN capsule) were evaluated for their predictive value for non-SN involvement.ResultsNon-SN metastases were found in 136/357 cases (38%). Microanatomic location and penetrative depth of SN metastases were significant predictors for non-SN involvement (<0.001); limited penetrative depth was associated with a low frequency of non-SN involvement with a minimal of 10%.ConclusionsMicroanatomic location and penetrative depth of breast cancer SN metastases predict non-SN involvement. However, based on these features no subgroup of patients could be selected with less than 10% non-SN involvement.

Expression of Ki-67 in the uterus during various stages of the estrous cycle in rats

Rats have an average estrous cycle of 4–5 days. There are four phases (proestrus, estrus, metesterus, and diestrus) in the estrous cycle in rodents. Histologic staging of the rodent estrous cycle is challenging and requires expertise. Thus, utilizing additional parameters such as cellular proliferation of the various components of the uterine microanatomy may assist with this process. Having an alternative method by which a pathologist can correctly identify the stages of the rodent estrous cycle would be valuable to the assessment and interpretation of safety studies for new drug candidates. This study was performed to investigate the microanatomic location of the uterine proliferative activity by image analysis and immunohistochemistry using Ki-67, a well-established marker of proliferating cells. Each stage of the rodent estrous cycle exhibited a different pattern of cellular proliferation. During proestrus, the lowest degree of cellular proliferation occurred in the glandular epithelial cells and the highest occurred in the myometrial cells. In estrus, lower levels of cellular proliferation were seen in the luminal and glandular epithelial cells, while a higher rate of proliferation occurred in myometrial cells followed by the stromal cells. At the metestrus stage, the highest cellular proliferation occurred in stromal and myometrial cells, while lesser proliferation was observed in luminal and glandular epithelial cells. This work demonstrates that in the rodent uterus there are cyclic changes in cellular proliferation in specific microanatomic uterine locations which can aid in the staging of the estrous cycle.

Insulin, leptin, and adiponectin receptors in colon: regulation relative to differing body adiposity independent of diet and in response to dimethylhydrazine

Obesity has recently become a focus of research to elucidate diet and lifestyle factors as important risk factors for colon cancer. Altered levels of insulin, leptin, and adiponectin have been identified as potential candidates increasing colon cancer risk within the prevailing obesogenic environment. There has been considerable research to characterize signaling via these hormones in the brain, liver, and adipose tissue; however, very little is known of their emerging role in peripheral signaling, particularly in epithelial tissues. This study profiles insulin, leptin, and adipokine receptors in the rat colon, revealing novel microanatomical location of these receptors and thereby supporting a potential role in regulating colonic tissue. Potential involvement of insulin, leptin, and adiponectin receptors in increased risk of colon cancer was investigated using Sprague-Dawley rats, either resistant or susceptible to diet-induced obesity. Regulation of insulin, leptin, and adiponectin receptors as a consequence of differing levels of adiposity was assessed regionally in the colon in response to treatment with the chemical carcinogen 1,2-dimethylhydrazine (DMH). However, significantly increased fat mass, increased levels of plasma insulin, leptin, and triglycerides, previously associated with an increased risk of colon cancer, were not associated with promotion of precancerous lesions in the experimental rats or deregulation of insulin, leptin, or adiponectin receptors. These findings do not support a direct link between the deregulation of insulin and adipokine levels observed in obese rats and an increased risk of colon carcinogenesis.

Pulmonary and Cardiorenal Cyclooxygenase-1 (COX-1), -2 (COX-2), and Microsomal Prostaglandin E Synthase-1 (mPGES-1) and -2 (mPGES-2) Expression in a Hypertension Model

Hypertensive mice that express the human renin and angiotensinogen genes are used as a model for human hypertension because they develop hypertension secondary to increased renin-angiotensin system activity. Our study investigated the cellular localization and distribution of COX-1, COX-2, mPGES-1, and mPGES-2 in organ tissues from a mouse model of human hypertension. Male (n = 15) and female (n = 15) double transgenic mice (h-Ang 204/1 h-Ren 9) were used in the study. Lung, kidney, and heart tissues were obtained from mice at necropsy and fixed in 10% neutral buffered formalin followed by embedding in paraffin wax. Cut sections were stained immunohistochemically with antibodies to COX-1, COX-2, mPGES-1, and mPGES-2 and analyzed by light microscopy. Renal expression of COX-1 was the highest in the distal convoluted tubules, cortical collecting ducts, and medullary collecting ducts; while proximal convoluted tubules lacked COX-1 expression. Bronchial and bronchiolar epithelial cells, alveolar macrophages, and cardiac vascular endothelial cells also had strong COX-1 expression, with other renal, pulmonary, or cardiac microanatomic locations having mild-to-moderate expression. mPGES-2 expression was strong in the bronchial and bronchiolar epithelial cells, mild to moderate in various renal microanatomic locations, and absent in cardiac tissues. COX-2 expression was strong in the proximal and distal convoluted tubules, alveolar macrophages, and bronchial and bronchiolar epithelial cells. Marked mPGES-1 was present only in bronchial and bronchiolar epithelial cells; while mild-to-moderate expression was present in other pulmonary, renal, or cardiac microanatomic locations. Expression of these molecules was similar between males and females. Our work suggests that in hypertensive mice, there are (a) significant microanatomic variations in the pulmonary, renal, and cardiac distribution and cellular localization of COX-1, COX-2, mPGES-1, and mPGES-2, and (b) no differences in expression between genders.

Lymphocyte‐independent connective tissue mast cells populate murine synovium

Mast cells (MCs) are a heterogeneous population of tissue-resident bone marrow-derived cells; distinct MC subpopulations are situated at specific microanatomic locations. The phenotype of the murine synovial MC remains undefined. Since MCs have been implicated in the pathogenesis of inflammatory arthritis, we sought to define the phenotype of the murine synovial MC population in normal and arthritic joints. We also examined the contribution of lymphocytes to synovial MC physiology. The MC phenotype in healthy and K/BxN serum transfer-induced arthritic synovial tissue was defined using immunohistochemical staining of prototypic MC-specific proteases (murine MC proteases [mMCP] 1, 2, 4, 5, 6, and 7) (chymases and tryptases). MC numbers and density were determined by histomorphometry in healthy and arthritic synovia. The lymphocyte contribution to MC populations was assessed using RAG-null mice. We found that synovial MCs display a connective tissue mast cell (CTMC) phenotype in both normal and arthritic synovial tissue, which expresses mMCP-4, -5, -6, and -7, but not mMCP-1 or mMCP-2. In addition, MC hyperplasia was seen in the arthritic synovium. In RAG-null mice, the phenotype and degree of MC hyperplasia were identical to those observed in normal mice with and without arthritis. Furthermore, in contrast to skin CTMCs, all synovial MCs expressed mMCP-6, demonstrating discrete differences between synovial CTMCs and other anatomic CTMC populations. Our findings demonstrate that the murine synovial MC population is composed of lymphocyte-independent CTMCs and identify arthritic synovium as a model system by which to gain insight into the poorly understood physiology of CTMCs in chronic inflammation.

Complex regulation of mucosal pentraxin ( Mptx) revealed by discrete micro-anatomical locations in colon

Recently a mucosal pentraxin, Mptx, regulated by heme and calcium was reported in rat gut mucosal scrapings using microarray strategies. Considering the heterogeneity of gut mucosa scrapings and the widespread use of the rat as a model to study colon pathologies this study was undertaken to generate detailed mapping of micro-anatomical locations of Mptx and gain further insight into potential functions of this mucosal pentraxin in rat colon. Differential regulation was also examined in colon from different rat strains and rat models of oxidative stress and in pre-cancerous colon tissue. Different regional patterns of expression and discrete localisation in epithelial cells within transverse and distal colon crypts and an absence of expression in proximal colon were confirmed by regional PCR analysis and in situ hybridisation studies of colon. This study demonstrates that consideration of regional differences in Mptx gene expression and micro-anatomical location is necessary in the interpretation and deciphering of its regulation in colon.

Novel sites of cytosolic glutathione peroxidase expression in colon

Glutathione peroxidases (Gpx) are important moderators of oxidative stress that is implicated in the pathogenesis of numerous diseases including colon cancer. Previous studies report limited examinations of cytosolic glutathione peroxidase location of expression in colon tissue. This study reports evidence of both common sites of Gpx1 and Gpx2 expression in rat colon and sites that are exclusive to each isoform. Semi-quantitative PCR performed previously demonstrated RNA expression of Gpx1 and Gpx2 in proximal, transverse and distal colon. Mapping the distribution throughout the entire colon has revealed specific, novel sites of glutathione peroxidase expression in colon lymphatic tissue. In situ hybridisation and immunohistochemistry confirmed micro-anatomical location of Gpx1 within lymphatic tissue and the lamina propria, sub-mucosa, muscularis and serosa, but not the lumenal epithelium. In situ hybridisation and immunohistochemistry were consistent with reports of microanatomical location of Gpx2 in the lumenal epithelium. Novel sites of Gpx2 expression were also observed in lymphatic tissue. Immunolocalisation in the vicinity of aberrant crypt foci was also examined to further investigate the link between glutathione peroxidases and colon cancer. This did not reveal significant abnormalities, nor did measurement of cytosolic glutathione peroxidase activity or gene expression in colon tissue from rats treated with the colontropic chemical, 1,2-dimethylhydrazine. These results support the potential for Gpx1 and Gpx2 redundancy in lymphatic tissue, but not in epithelial cells of the colon crypt or in the lamina propria, sub-mucosa, muscularis or serosa.