Microanatomic Location Research Articles

Microanatomical Labeling of Germinal Center Structures for Flow Cytometry Using Photoactivation.

Germinal centers (GCs) are inducible microanatomical structures required for the generation of high-affinity antibodies. Migration of B and T cells within and into/out of GCs plays a key role in the evolutionary process that underlies affinity maturation, and thus microanatomical location of cells is an important variable when studying GC processes. We describe a protocol in which in situ photoactivation by multiphoton microscopy can be used to add microanatomical information to flow cytometry, allowing for identification and isolation of GC cells based on their location. Cells in different microanatomical compartments can then be sorted and analyzed for surface marker and mRNA expression.

Microanatomical Location of Hyaluronic Acid Gel Following Injection of the Temporal Hollows.

To examine the microanatomical location of hyaluronic acid gel injected within the temporal hollows of cadaver specimens. The temporal hollows were injected subcutaneously with hyaluronic acid gel in 6 fresh frozen human cadaver hemifaces. Temporal soft tissues were dissected to a preperiosteal plane and fixated in 95% alcohol. A soft tissue section extending from skin to temporal bone was obtained for each specimen. Histologic examination was performed using hematoxylin and eosin stain. In 5 of 6 specimens, at least 95% of the hyaluronic acid was located within the subcutaneous fat. In 1 of 6 specimens, approximately 35% of the material was located within the subcutaneous fat and 60% was located within the superficial temporal fascia. Two specimens had 5% located within the temporalis muscle. In 1 specimen, hyaluronic acid was found to encompass a superficial muscular artery within the superficial temporal fascia. This study elucidates the location of hyaluronic acid gel after subcutaneous injection within the temporal hollow. Histology confirmed consistent placement of the gel within the subcutaneous tissues, but it also showed that injection in this region may produce unintended deeper location of filler, and a significant perivascular collection of the material. The proximity of dense temporal fascial and muscle arterial networks in this region may pose risk for perivascular injection and associated complications.

Suppression of Long-Lived Humoral Immunity Following Borrelia burgdorferi Infection.

Lyme Disease caused by infection with Borrelia burgdorferi is an emerging infectious disease and already by far the most common vector-borne disease in the U.S. Similar to many other infections, infection with B. burgdorferi results in strong antibody response induction, which can be used clinically as a diagnostic measure of prior exposure. However, clinical studies have shown a sometimes-precipitous decline of such antibodies shortly following antibiotic treatment, revealing a potential deficit in the host’s ability to induce and/or maintain long-term protective antibodies. This is further supported by reports of frequent repeat infections with B. burgdorferi in endemic areas. The mechanisms underlying such a lack of long-term humoral immunity, however, remain unknown. We show here that B. burgdorferi infected mice show a similar rapid disappearance of Borrelia-specific antibodies after infection and subsequent antibiotic treatment. This failure was associated with development of only short-lived germinal centers, micro-anatomical locations from which long-lived immunity originates. These showed structural abnormalities and failed to induce memory B cells and long-lived plasma cells for months after the infection, rendering the mice susceptible to reinfection with the same strain of B. burgdorferi. The inability to induce long-lived immune responses was not due to the particular nature of the immunogenic antigens of B. burgdorferi, as antibodies to both T-dependent and T-independent Borrelia antigens lacked longevity and B cell memory induction. Furthermore, influenza immunization administered at the time of Borrelia infection also failed to induce robust antibody responses, dramatically reducing the protective antiviral capacity of the humoral response. Collectively, these studies show that B. burgdorferi-infection results in targeted and temporary immunosuppression of the host and bring new insight into the mechanisms underlying the failure to develop long-term immunity to this emerging disease threat.

Endemic and highly prevalent amyloid A amyloidosis in endangered island foxes (Urocyon littoralis) (1139.16)

Critically endangered island foxes develop a systemic amyloidosis with an extraordinarily high prevalence of 32% (mean), and exceeding 40% in some subspecies. Amyloidosis has been recognized in foxes for 15 years. The organs most affected by amyloid are kidney, spleen, vessels, heart, adrenal gland, tongue, oropharynx, and skin. There was an increased risk of amyloidosis found in adults, the San Clemente island subspecies, captively housed foxes, and foxes with neoplasia. In amyloid‐rich tissue, the dominant insoluble protein was a 12‐kDa protein labeled by serum amyloid A (SAA) antibodies that were generated against canine SAA. Mass spectrometry on insoluble proteins from 9 amyloid‐laden samples revealed SAA as the dominant protein, which was not present in control samples. The SAA protein was composed of 111 amino acids and consisted of multiple isoforms, including isoforms unique amongst canids. SAA amyloidosis is typically associated with prolonged elevation of SAA in serum, and island foxes have a variety of chronic inflammatory diseases that may lead to elevated SAA levels. We found that conformationally sensitive amyloid binding probes known as luminescent conjugated polymers bind the island fox SAA amyloid and emit different spectra within different microanatomical locations in the kidney, suggesting there may be conformational or compositional differences in amyloid deposits.

Comparison of Sentinel Lymph Node Micrometastatic Tumor Burden Measurements in Melanoma

Multiple methods have been proposed to classify the micrometastatic tumor burden in sentinel lymph nodes (SLN) for melanoma. The purpose of this study was to determine the classification scheme that best predicts nonsentinel node (NSN) metastasis, disease-free survival (DFS), and overall survival (OS). A single reviewer reanalyzed tumor-positive SLN from a multicenter, prospective clinical trial of patients with melanoma ≥ 1.0 mm Breslow thickness who underwent SLN biopsy. The following micrometastatic disease burden measurements were recorded: Starz classification, Dewar classification (microanatomic location), maximum diameter of the largest focus of metastasis, maximum tumor area, and sum of all diameters. Univariate and multivariate models and Kaplan-Meier analysis were used to evaluate each classification system. We reviewed 204 tumor-positive SLNs from 157 patients. On univariate analysis, all criteria except Starz classification were statistically significant risk factors for NSN metastasis. On multivariate analysis, including Breslow thickness, ulceration, age, sex, and NSN status, maximum diameter (using a cut-off of 3 mm) was the only classification system that was an independent risk factor predicting DFS (hazard ratio 2.31, p = 0.0181) and OS (hazard ratio 3.53, p = 0.0005). By Kaplan-Meier analysis, DFS and OS were significantly different among groups using maximum diameter cut-offs of 1 and 3 mm. Maximum tumor diameter outperformed other measurements of metastatic tumor burden, including microanatomic tumor location (Dewar classification), Starz classification, maximum tumor area, and sum of all diameters for prediction of survival. Maximum tumor diameter is a simple method of assessing micrometastatic tumor burden that should be reported routinely.

Perspectives on the immunologic microenvironment of astrocytomas

BackgroundThe microenvironment of astrocytomas includes infiltrative inflammatory cells that are dynamic in nature, possibly reflecting tumor biology. We evaluated the inflammatory cell infiltrate in astrocytic tumors aiming for a better understanding of their immunobiology.MethodsImmunohistochemical expression of CD68, CD3, and CD20 was investigated in 21 glioblastomas, 21 anaplastic astrocytomas, 13 diffuse astrocytomas, and 18 pilocytic astrocytomas. The inflammatory infiltrate was classified based on microanatomic location as perivascular and intratumoral, and subsequently graded semiquantitatively.ResultsPerivascularly, CD68-positive infiltrate was noted in 71.4% of glioblastomas compared with 14.3% of anaplastic astrocytomas (P = 0.0001), 7.7% of diffuse astrocytomas (P = 0.0001), and 33.3% of pilocytic astrocytomas (P = 0.017). Intratumorally, 85.7% of glioblastomas exhibited CD68-positive infiltrate compared with 42.9% of anaplastic astrocytomas (P = 0.004), 38.5% of diffuse astrocytomas (P = 0.008), and 33.3% of pilocytic astrocytomas (P = 0.001). Among diffusely infiltrating astrocytomas, intratumoral CD3-positive infiltrate was only associated with glioblastoma. A CD20-positive infiltrate was only detected in the perivascular space of a single case of diffuse astrocytoma.ConclusionThese data indicate a distinct immune profile in the glioblastoma microenvironment primarily related to the prevalence of macrophages. Thus, novel glioblastoma therapies should address this key CD68-positive population and its possible role in generating an antitumor immune response.

On the Brain of a Crustacean: A Morphological Analysis of CaMKII Expression and Its Relation to Sensory and Motor Pathways

Calcium/calmodulin kinase II (CaMKII) is a Ca2+-activated enzyme that is abundant in vertebrate and invertebrate brains. However, its characterization is poorly addressed in the nervous system of crustaceans, and, to our knowledge, no studies have determined the microanatomical location of CaMKII in a crustacean species. In this study, we found labeling of CaMKII in the eyestalk and brain of the prawn Macrobrachium acanthurus, by means of immunohistochemistry and Western blotting. Antibodies against neuron (ß tubulin III), glutamate receptor (GluA1), and FMRFamide were used in order to further characterize the CaMKII-labeled cells in the brain. In the eyestalk, strong labeling with CaMKII was observed in the photoreceptors. These cells, especially in the rhabdom, were also reactive to anti-ß tubulin III, whereas the pigment cells were labeled with anti-CaMKII. GluA1 co-located with CaMKII in the photoreceptors. Also, CaMKII appeared in the same sites as FMRFamide in the deutocerebrum, including the olfactory lobe, and in the tritocerebrum, specifically in the antennular neuropil, indicating that the synaptic areas in these regions may be related to sensory-motor processing. In the brain, the identification of cells and regions that express CaMKII contributes to the understanding of the processing of neural connections and the modulating role of CaMKII in decapod crustaceans.

The Prognostic Value of Minimally Involved Melanoma Sentinel Lymph Nodes

Background: Sentinel node (SLN) status is the most important prognostic factor for early-stage melanoma patients. It will influence follow-up and may change therapy. Positive SLNs present different degrees of involvement so that subgroups of patients may have minimal SLN invasion. The aim of this study was to evaluate survival in subgroups with minimally involved SLNs and to compare them to negative SLN patients. Method: SLN biopsy was performed in 499 consecutive clinically N0 patients between 1997 and 2008. Following updated recommendations from the Melanoma Group of the European Organization of Research and Treatment of Cancer, degrees of SLN involvement were fully reassessed for two anatomopathological parameters: tumour burden according to Rotterdam criteria ( 1.0 mm) and microanatomic location according to Dewar (subcapsular, combined subcapsular and parenchymal, parenchymal, multifocal, or extensive). Minimally involved SLNs were defined as those with tumor burden <0.1 mm and/or subcapsular metastasis location. Kaplan-Meier and multivariable logistic regression analyses were performed. Results: Out of 499 clinically N0 patients, positive SLNs were found in 123 patients (24.7 percent). With a median follow-up of 52 months (range: 9 - 146), five-year disease-free (DFS), disease-specific survival (DSS) and overall survival (OS) were 88.1, 93.9 and 89.9 percent for negative SLN patients, respectively. In minimally involved SLNs, there were 21 with tumour burden <0.1 mm, and 52 with subcapsular metastasis. Five-year DFS, DSS and OS in these sub-groups were 79.6, 86.6 and 86.6 percent, then 57.3, 69.8 and 67.8 percent respectively. DFS univariable analysis of these sub-groups compared to negative SLNs showed: (HR1.89, 95 percent CI 0.75 - 4.79; p 0.175) and (HR 3.92, 95 percent CI 2.29 - 6.71; p < 0.0001) respectively. Minimally involved sub-groups were not predictive for NSLN negativity. Conclusion: Rotterdam’s tumour burden stratification is an easy and useful prognostic factor of melanoma survival. There was a trend showing that patients with SLN tumour burden <0.1 mm have a lower survival compared to SLN negative patients. One might suggest that patients with minimally involved SLNs may not be managed similarly to negative SLN patients. Subcapsular metastasis subgroup according to the microanatomic location has statistically significant worst survival.

Differentiation of germinal center B cells and follicular helper T cells as viewed by tracking Bcl6 expression dynamics

Development of germinal center (GC) B cells and follicular helper T (Tfh) cells requires the transcription factor B-cell lymphoma 6 (Bcl6). Expression of Bcl6 in B cells and helper T cells is regulated by complex signals including those generated through their antigen-specific interactions, which take place in various microenvironments depending on their activation/differentiation states. In the last several years, it has become possible to detect Bcl6 protein in individual B cells and T cells by intracellular staining with the newly developed antibodies or by using the reporter mice. Experiments using these reagents have started to clarify microanatomical location of early Bcl6 upregulation in B cells and T cells, and contributed to reveal the dispensability and indispensability of B cells in the early and late phase of Tfh differentiation. They also started to reveal the diversity, plasticity, and/or instability of Tfh cells. We summarize the recent findings made by tracking Bcl6 expression together with the updated knowledge about dynamics of antigen-engaged B cells and helper T cells and discuss them in relation to possible signaling requirements for the development of GC B cells and Tfh cells.

EORTC Melanoma Group achievements

Since its inception in 1969, the EORTC Melanoma Group has employed a multidisciplinary approach in the fight against melanoma and has registered significant achievements in many areas of melanoma treatment and research. The group showed that sentinel node (SN) tumor burden according to the Rotterdam Criteria and the microanatomic location were the most important prognostic factors for melanoma-specific survival and non-SN positivity in the completion lymph node dissection specimen. They demonstrated that extended schedule escalated dose temozolomide is feasible and has an acceptable safety profile. They also showed that the interferon-a targeted therapy should occur in a targeted patient population, and should probably not be offered to 70% of the patients that are currently being given this treatment. Through EORTC trial 18991, Sylatron™, pegylated interferon a-2b, for the treatment of melanoma patients with microscopic or gross nodal involvement within 84 days of definitive surgical resection including complete lymphadenectomy, was approved by the US FDA. The present article describes the achievements and future strategies of the Melanoma Group.

Age Dependent Alternations In Hematopoietic Stem Cell Niches

Abstract 2395Age associated decline in hematopoietic system is a well-documented process which encompasses a substantial expansion of the hematopoietic stem cell (HSC) pool and a decline of their competitive repopulating ability on a per cell basis. Expansion occurs predominantly in the myeloid biased HSC subset, subsequently leading to a global skewing of lineage differentiation from lymphopoiesis toward myelopoiesis. These age-related effects are known to partially result from cell-intrinsic alterations of HSC, which include DNA damage accumulation, oxidative stress and activation of tumor suppressor pathways. HSCs exist in specialized microanatomical locations within the bone marrow (BM), where they receive critical regulatory cues that control HSC homeostasis. While the cell-autonomous effects of HSC aging have been extensively studied, little is known on how aging of the surrounding microenvironment may contribute to the age-associated alterations of HSC function, and thus the focus of our study.Here, we have employed novel imaging approaches to analyze age-related changes of the BM microenvironment, and how these affect the global spatial distribution of HSPC-enriched subsets in the BM parenchyma. Our preliminary studies suggest during aging leads to a profound disruption of the BM microvascular compartment. By using imaging cytometry of two-dimensional (2D) sections, as well as confocal microscopy of 3D-BM slices, we demonstrate that the characteristic sinusoidal network found in young mice, degenerated with aging into a highly disorganized and expanded vascular bed, in which expression of the arterial marker Sca-1 was strongly upregulated. Flow cytometry analysis further revealed a 3–4 fold increase of CD45-Ter119-CD31+ Sca-1+cells in 22–24 month old mice. We next investigated how the changes in vasculature network impact HSC distribution. We employed imaging cytometry to track the presence in BM sections of a population of Lineage-Cd48-kit+ cells, 60% of which are long term HSC. Our preliminary results indicate that, in aged mice, HSPC-accumulation in endosteal zones (<100μm from endosteal surfaces) remains unchanged. Of note, consistent with the increase in vascularization, HSPCs display a significantly increased tendency to directly associate to BM microvessels in 22–24month old mice. We are currently investigating at the molecular level how altered vasculature influence HSC aging which would enable us to enhance the regenerative properties of the aged stem cells. Disclosures:Wagers:iPierian, Inc.: Membership on an entity's Board of Directors or advisory committees.

Prediction of additional lymph node positivity and clinical outcome of micrometastases in sentinel lymph nodes in cutaneous melanoma: A multi-institutional study of 450 patients in Japan

Various microscopic classifications of metastatic sentinel lymph nodes (SLN) have been reported along with predictors of additional lymph node positivity and their correlations with the prognosis. The purpose of this study was to re-evaluate these classifications in the Japanese population. We selected the following three classifications, based on the procedural simplicity of the measurements: maximum diameter (maximum diameter of the largest tumor lesion in the SLN; <0.1, 0.1-1.0, >1.0 mm), invasion depth (depth of tumor invasion measured from the capsule in the SLN; SI ≤ 0.3 mm, SII >0.3 to ≤ 1.0 mm, SIII >1.0 mm), and microanatomic location (microanatomic location of the tumor deposits within the SLN; "subcapsular", "parenchymal", "combined", "multifocal", "extensive"). A retrospective study, using prescribed survey forms, was carried out. Among the 450 patients, including the 149 cases with SLN metastasis, an additional lymph node positivity rate of 0% could be predicted only in patients with a maximum diameter category of less than 0.1 mm. As compared with that in the SLN metastasis-negative cases, however, the prognosis was poorer in cases with SLN metastasis, even those with lesions falling under the maximum diameter category of less than 0.1 mm, invasion depth category of SI (≤ 0.3 mm) and microanatomic location category of subcapsular. The prognosis is particularly poor for the microanatomic location category of extensive, which should thus be regarded as a macrometastasis. A prospective study with standardized procedures, including pathological evaluation, is needed in order to confirm our conclusion.

Prognostic impact of isolated tumor cells in breast cancer axillary nodes: single tumor cell(s) versus tumor cell cluster(s) and microanatomic location

In breast cancer, it has been shown that pN0(i+) and pN1mi have a comparable negative impact on disease-free survival, compared with pN0. However, pN0(i+) is considered to be a heterogeneous group. We determined the effect of metastatic size and microanatomic location within the pN0(i+) group on breast cancer recurrence. We included all Dutch breast cancer patients diagnosed in 1998-2005 with favorable primary tumor characteristics and a final nodal status of pN0(i+). For this analysis, only patients without adjuvant systemic therapy were eligible (n = 513). Presence of single tumor cells versus cell clusters, metastatic size and microanatomic location were recorded. Primary endpoint was disease-free survival. Analyses were adjusted for age at diagnosis, tumor size, tumor grade, axillary treatment and hormone receptor status. The 5-year disease-free survival of patients with single tumor cell(s) (n = 93) was 78.6% and with tumor cell cluster(s) (n = 404) 77.1%. The hazard ratio for disease events was 1.05 (95% CI 0.63-1.76) for cell cluster(s) compared with single cell(s). In a Cox regression model, doubling of metastatic tumor size corresponded to a hazard ratio of 1.21 (95% CI 1.02-1.43). The adjusted hazard ratio was 0.90 (95% CI 0.54-1.50) for parenchymal (n = 112) versus sinusoidal location (n = 395). Single tumor cells 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.

Immune cell infiltrate differences in pilocytic astrocytoma and glioblastoma: evidence of distinct immunological microenvironments that reflect tumor biology

The tumor microenvironment in astrocytomas is composed of a variety of cell types, including infiltrative inflammatory cells that are dynamic in nature, potentially reflecting tumor biology. In this paper the authors demonstrate that characterization of the intratumoral inflammatory infiltrate can distinguish high-grade glioblastoma from low-grade pilocytic astrocytoma. Tumor specimens from ninety-one patients with either glioblastoma or pilocytic astrocytoma were analyzed at the University of California, San Francisco. A systematic neuropathology analysis was performed. All tissue was collected at the time of the initial surgery prior to adjuvant treatment. Immune cell infiltrate not associated with necrosis or hemorrhage was analyzed on serial 4-μm sections. Analysis was performed for 10 consecutive hpfs and in 3 separate regions (total 30 × 0.237 mm(2)). Using immunohistochemistry for markers of infiltrating cytotoxic T cells (CD8), natural killer cells (CD56), and macrophages (CD68), the inflammatory infiltrates in these tumors were graded quantitatively and classified based on microanatomical location (perivascular vs intratumoral). Control markers included CD3, CD20, and human leukocyte antigen. Glioblastomas exhibited significantly higher perivascular (CD8) T-cell infiltration than pilocytic astrocytomas (62% vs 29%, p = 0.0005). Perivascular (49%) and intratumoral (89%; p = 0.004) CD56-positive cells were more commonly associated with glioblastoma. The CD68-positive cells also were more prevalent in the perivascular and intratumoral space in glioblastoma. In the intratumoral space, all glioblastomas exhibited CD68-positive cells compared with 86% of pilocytic astrocytomas (p = 0.0014). Perivascularly, CD68-positive infiltrate was also more prevalent in glioblastoma when compared with pilocytic astrocytoma (97% vs 86%, respectively; p = 0.0003). The CD3-positive, CD20-positive, and human leukocyte antigen-positive infiltrates did not differ between glioblastoma and pilocytic astrocytoma. This analysis suggests a significantly distinct immune profile in the microenvironment of high-grade glioblastoma versus low-grade pilocytic astrocytoma. This difference in tumor microenvironment may reflect an important difference in the tumor biology of glioblastoma.

Minireview: The Stem Cell Next Door: Skeletal and Hematopoietic Stem Cell “Niches” in Bone

Long known to be home to hematopoietic stem cells (HSC), the bone/bone marrow organ and its cellular components are directly implicated in regulating hematopoiesis and HSC function. Over the past few years, advances on the identity of HSC "niche" cells have brought into focus the role of cells of osteogenic lineage and of marrow microvessels. At the same time, the identity of self-renewing multipotent skeletal progenitors (skeletal stem cells, also known as mesenchymal stem cells) has also been more precisely defined, along with the recognition of their own microvascular niche. The two sets of evidence converge in delineating a picture in which two kinds of stem cells share an identical microanatomical location in the bone/bone marrow organ. This opens a new view on the manner in which the skeleton and hematopoiesis can cross-regulate via interacting stem cells but also a novel view of our general concept of stem cell niches.

Functional genomics unique to week 20 post wounding in the deep cone/fat dome of the Duroc/Yorkshire porcine model of fibroproliferative scarring.

BackgroundHypertrophic scar was first described over 100 years ago; PubMed has more than 1,000 references on the topic. Nevertheless prevention and treatment remains poor, because 1) there has been no validated animal model; 2) human scar tissue, which is impossible to obtain in a controlled manner, has been the only source for study; 3) tissues typically have been homogenized, mixing cell populations; and 4) gene-by-gene studies are incomplete.Methodology/Principal FindingsWe have assembled a system that overcomes these barriers and permits the study of genome-wide gene expression in microanatomical locations, in shallow and deep partial-thickness wounds, and pigmented and non-pigmented skin, using the Duroc(pigmented fibroproliferative)/Yorkshire(non-pigmented non-fibroproliferative) porcine model. We used this system to obtain the differential transcriptome at 1, 2, 3, 12 and 20 weeks post wounding. It is not clear when fibroproliferation begins, but it is fully developed in humans and the Duroc breed at 20 weeks. Therefore we obtained the derivative functional genomics unique to 20 weeks post wounding. We also obtained long-term, forty-six week follow-up with the model.Conclusions/Significance1) The scars are still thick at forty-six weeks post wounding further validating the model. 2) The differential transcriptome provides new insights into the fibroproliferative process as several genes thought fundamental to fibroproliferation are absent and others differentially expressed are newly implicated. 3) The findings in the derivative functional genomics support old concepts, which further validates the model, and suggests new avenues for reductionist exploration. In the future, these findings will be searched for directed networks likely involved in cutaneous fibroproliferation. These clues may lead to a better understanding of the systems biology of cutaneous fibroproliferation, and ultimately prevention and treatment of hypertrophic scarring.

Optical marking of migrating lymphocytes by two-photon photoconversion for single cell tracking and fate mapping (104.22)

Abstract We present a method for localised two-photon photoconversion of fluorescent cells within distinct microanatomical compartments in intact lymph nodes. This method allows efficient photoconversion of whole cell populations as well as of single cells while preserving cell viability and motility. Moreover, the combination of high-speed image acquisition and real-time display permits us to target and lock-on to individual cells for photoconversion as they migrate within the lymph node. Optical cell marking makes it possible to differentiate otherwise homogenous responding cells based on their microanatomical location. It also enables discontinuous in vivo tracking of cell fates to distal sites after extended time intervals.

Value of micromorphometric criteria of sentinel lymph node metastases in predicting further nonsentinel lymph node metastases in patients with melanoma

Patients with metastases in the sentinel node (SN) are advised to undergo complete lymph node dissection, although the majority of them will have no further metastatic disease. Some of these patients undergo unnecessary surgery. In this study, we tried to predict the likelihood of further non-SN metastases on the basis of earlier published micromorphometric classifications of SN metastases. Metastases in the SN were re-evaluated on the basis of the microanatomic location of the lesions according to the Dewar's criteria, the S-classification of SN, and tumor burden in accordance with the Rotterdam criteria. The results of these classifications were correlated with the presence of further non-SN metastases. Specimens of 124 positive-SN basins and subsequent complete lymph node dissection were investigated. Further metastases in non-SNs were found in 30 lymph node basins (24.2%). All of the above-mentioned classification systems were significantly correlated with non-SN tumor status. Especially, in patients with SN metastases in subcapsular location, a maximum depth of invasion of less than 0.3 mm (stage I according to the S-classification) or metastases of less than 0.1 mm in diameter had a very low probability of further non-SN metastases (0-5%). The validity of earlier published classifications of SN metastases-based on the micromorphometric criteria in predicting non-SN status was confirmed. Especially, in patients with subcapsular metastases, SI stage metastases or metastases of less than 0.1 mm had a very low risk of further non-SN metastases.

Mast Cell distribution and micro-anatomical location after bilateral uterine artery ligation in mature rabbits

To determine the effect of bilateral uterine artery ligation (BUAL) on mast cell (MC) distribution, 24 mature female rabbits were studied. The rabbits were ovariohysterectomized on day 23, 43 or 63 following BUAL. Histological sections were stained with toluidine blue to determine the MC distribution. In rabbits that had undergone BUAL, MCs were present extensively in the helium of the treated ovaries, in dense groups close to the blood vessels. In the control group, MCs were observed at a medium density around the blood vessels and the number of MCs in the reproductive tract was significantly (P ? 0.01) lower than in the test groups. This study demonstrates that, after BUAL, the distribution and numbers of MCs differ between different parts of the reproductive system.

Ontogeny of pulmonary cyclooxygenase-1 (COX-1) and -2 (COX-2)

Prostaglandins synthesized by enzymatic reactions such as cyclooxygenases have been implicated in lung pathophysiology. The goal of this study was to delineate the pulmonary ontogeny of cyclooxygenase enzymes (COX-1 and COX-2) immunohistochemical expression and cellular localization in various microanatomic locations of lungs from pre-term, term, and post-natal lambs. Lung tissues were obtained at 115 and 130 days of gestation from pre-term lambs, 145 days (term; complete gestation), and 15 days post-natally. No significant differences were seen in lung COX-1 expression at various microanatomic locations during pre-term, term, or postnatally. Moderate to strong COX-1 expression was present in macrophages, alveolar septa, bronchial smooth muscle cells, bronchiolar smooth muscle cells, vascular endothelial cells, and vascular smooth muscle cells. Minimal COX-1 expression was present in bronchial and bronchiolar epithelial cells. Most microanatomic locations lacked COX-2 expression with the exception of weak expression that was present in bronchial and bronchiolar epithelial cells at 145 days of full gestation and 15 days post-natally. This work suggests that: (a) COX-1 is constitutively expressed in lungs from pre-term, term, and post-natal lambs in various microanatomic pulmonary locations, (b) there is differential expression of COX-1 and COX-2 in the developing lung, and (c) COX-2 does not appear to play a role in lung fetal development, at least in neonatal lambs.