Microanatomic Location Research Articles

The microanatomic location of metastatic melanoma in sentinel lymph nodes predicts nonsentinel lymph node involvement.

Sentinel node biopsy is now widely accepted as the most accurate prognostic indicator in melanoma, and is important in guiding management of patients with clinical stage I or II disease. Patients with a positive sentinel node have conventionally undergone completion lymphadenectomy (CLND) of the involved basin, but only 20% have involvement beyond the sentinel node, suggesting that CLND may be unnecessary for the other 80% of patients. This study seeks to identify criteria that might be used to be more restrictive in selecting those who should undergo CLND. A total of 146 patients were identified who had had a positive sentinel node biopsy for malignant melanoma. Their sentinel nodes and lymphadenectomy specimens were re-evaluated pathologically. The metastatic melanoma in each sentinel node was assessed according to its microanatomic location within the node (subcapsular, combined subcapsular and parenchymal, parenchymal, multifocal, or extensive), and this was correlated with the presence of involved nonsentinel nodes in the CLND. The depth of the metastases from the sentinel node capsule was also recorded. The metastatic deposits in the sentinel node were subcapsular in 26.0% of patients. None of these patients had any nonsentinel nodes involved on CLND. In the patients whose sentinel node metastases had a different microanatomic location, the rate of nonsentinel node involvement was 22.2% overall. The microanatomic location of metastases within sentinel nodes predicts nonsentinel lymph node involvement. In patients with only subcapsular deposits in the sentinel node, it is possible that CLND could safely be avoided.

Nanomechanical properties and mineral concentration in articular calcified cartilage and subchondral bone.

We studied articular calcified cartilage (ACC) and the immediately subchondral bone (SCB) in normal and osteoarthritic human femoral heads. Thick slices of human normal reference post mortem (PM) and osteoarthritic (OA) femoral heads (age 55-89 years) were embedded in PMMA, micromilled, carbon coated and studied using quantitative backscattered electron (qBSE) imaging to determine variations in degree of mineralization. With exact microanatomical location, nanoindentation was performed on the same block faces in representative superior (more highly loaded) and medial regions of the joint surface. Using a partial unloading method, elastic modulus as a function of indenter penetration depth was determined using a spherical tipped diamond indenter. A pointed indenter was used to determine the tissue hardness in selected locations. The relationship between mineralization and indentation modulus was more distinct in ACC than in SCB, the latter having a higher matrix concentration with variable collagen orientation. In OA, the bulk of the measurements were coincident with those in the PM samples, although there was a greater range in the levels of mineralization and modulus in ACC. In OA, extremely hypermineralized ACC was found in ACC proper, especially in superior regions, and translocated into SCB and hyaline cartilage. The very highly mineralized cartilage fragments may function as a hard grinding abrasive, accelerating wear rates whether attached to or fragmented from the eburnated surfaces of OA ACC. Highly mineralized regions would also alter loading patterns and thereby contribute to further destruction of the joint tissues.

Combination of microdissection and microarray analysis to identify gene expression changes between differentially located tumour cells in breast cancer

Comparison of gene expression changes between cancer cells at the periphery and in the centre of breast cancers was performed using a combination of microdissection and microarray analysis. Cancer cells from the two areas were pooled separately from five patients with ductal carcinoma in situ and separately from five patients with frankly invasive cancer. Limited total RNA, 100-200 ng, from this microdissected tissue required use of the Atlas SMART trade mark Probe Amplification Kit to synthesize and amplify cDNA and make (33)P-labelled probes. Probes were then hybridized to Atlas Human Cancer 1.2 Arrays containing 1176 known genes. Triplicate analysis revealed that 22 genes changed their expression levels in the periphery relative to the central region: 15 upregulated and seven downregulated (arbitrary threshold of 1.5-fold or greater). Differences in RNA levels were confirmed by quantitative real-time PCR for two of the genes and by changes in protein levels, detected by immunohistochemistry, for a couple of representative gene products. Thus, changes in gene expression associated with variation in microanatomical location of neoplastic cells can be detected within even small developing tumour masses.

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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.

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Strong expression of the lymphoattractant C-X-C chemokine Mig is associated with heavy infiltration of T cells in human malignant melanoma.

Human malignant melanoma (MM) is a highly aggressive tumour which is particularly prone to specific local immune responses. To determine the microanatomical location and the species of chemokines possibly involved in the intricate control of cell migration and positioning of immune effector cells in primary and metastatic MM lesions, the expression of those chemokines with lymphocyte and/or macrophage chemoattractant properties was analysed by in situ hybridization. GROalpha (growth-related oncogene) and IL-8 (interleukin 8) were expressed at low levels by single melanoma cells, adjacent keratinocytes, and infiltrating leukocytes. In contrast, the lymphocyte-specific chemokine Mig (monokine induced by interferon-gamma) was strongly expressed by mononuclear cells (mainly macrophages) infiltrating the tumour margin in primary MM lesions, whereas expression was less intense in MM metastasis. IP-10 (interferon-gamma inducible protein 10) was expressed in the same loci at lower intensity. Marked infiltration of T cells was exclusively detected in those areas which exhibited strong Mig expression, whereas areas in the vicinity of tumour cells devoid of Mig expression were not infiltrated. In contrast to Mig, expression of MCP-1 (macrophage chemotactic protein-1) was weaker and mainly detected in lesional basal keratinocytes, occasionally at sites of macrophage infiltration, as well as in single melanoma cells. MIP-1alpha (macrophage inflammatory protein 1alpha) showed similar, albeit weaker expression compared with MCP-1. Other chemokines relevant for the recruitment of monocytes and lymphocytes, such as RANTES (regulated on activation, normal T cells expressed and secreted) and MIP-1beta, were barely detectable. In summary, the chemokine expression profiles support the notion that particularly in heavily infiltrated primary MM lesions, Mig and to a lesser extent IP-10 are important mediators of an IFN-gamma-dependent pathway. Due to their lymphoattractant properties and the known inhibitory effects on the tumour vasculature, both chemokines may be critical for the control of local melanoma tumour growth.

Heterogeneity of Mouse Spleen Dendritic Cells: In Vivo Phagocytic Activity, Expression of Macrophage Markers, and Subpopulation Turnover

In the normal mouse spleen, two distinct populations of dendritic cells (DC) are present that differ in microanatomical location. The major population of marginal DC is found in the "marginal zone bridging channels" and extends into the red pulp. The interdigitating cells (IDC) are localized in the T cell areas in the white pulp. The aim of the present study was to characterize these two splenic DC populations with regard to their phenotype, in vivo phagocytic function, and turnover. Both marginal DC and IDC are CD11c+ and CD13+, but only IDC are NLDC-145+ and CD8alpha+. Notably, both populations, when freshly isolated, express the macrophage markers F4/80, BM8, and Mac-1. To study the phagocytic capacity of these cells, we employed the macrophage "suicide" technique by injecting liposomes loaded with clodronate i.v. Marginal DC, but not IDC, were eliminated by this treatment. Phagocytosis of DiI-labeled liposomes by DC confirmed this finding. The two DC populations differed significantly with regard to their turnover rates, as studied in a transgenic mouse model of conditional depletion of DC populations with high turnover. In these mice, marginal DC were completely eliminated, but the IDC population remained virtually intact. From these data we conclude that the marginal DC population has a high turnover, in contrast to the IDC population. Taken together, the present results indicate that marginal DC and IDC represent two essentially distinct populations of DC in the mouse spleen. They differ not only in location, but also in phenotype, phagocytic ability, and turnover.

Some aspects of IL-8 pathophysiology III: chemokine interaction with endothelial cells

Chemokines have been convincingly implicated in driving leukocyte emigration in different inflammatory reactions. However, the cellular and molecular mechanisms of chemokine involvement in leukocyte emigration are not clear. We and others suggested that leukocyte adhesion to the endothelium and transmigration are induced by chemokines immobilized on the endothelial cell surface. This would require the presence of specific chemokine binding sites in this microanatomical location. Using an in situ binding assay we demonstrated the presence of binding sites for interleukin-8 (IL-8) and RANTES, but not monocyte inflammatory protein-1 alpha on the endothelium of postcapillary venules and small veins in human skin. In contrast, venules and veins in various anatomical locations showed dramatically differing IL-8 binding patterns. The subcellular distribution of IL-8 in the venular endothelial cells following its in vivo and ex vivo injections was studied by use of electron microscopy. Our results suggest that IL-8 was internalized by the endothelial cells, transported transcellularly via plasmalemmal vesicles, and released onto the luminal surface where it appeared located preferentially on tips of membrane protrusions. We were unable to study the endothelial IL-8 binding or transport in vitro because all the in vitro propagated endothelial cell lines and primary endothelial cells tested lacked IL-8 binding sites. This includes human umbilical vein endothelial cells (HUVECs), which also did not bind IL-8 in situ. However, HUVECs provided a satisfactory in vitro system to study the secretion of IL-8 by the endothelial cells. Two possible alternative pathways were described: secretion directly from the Golgi apparatus or following storage in Weibel-Palade bodies.

Measurement of the tissue distribution of immunoperoxidase staining with polyclonal anti‐BCG serum in lung granulomata of mice infected with Mycobacterium tuberculosis

Mice inoculated with Mycobacterium tuberculosis, strain H37Rv were used as a model of human tuberculosis. The microanatomical location of immunoperoxidase staining with a polyclonal anti-BCG serum was within macrophages and appeared granular rather than delineating whole bacilli. Immunoperoxidase staining appears to demonstrate degraded mycobacterial antigens from disrupted organisms and so reflects prior turnover of bacilli. On Ziehl-Neelsen staining, intact or almost intact bacilli are seen and so the extent of this form of staining reflects the current bacillary load. Both methods have limited sensitivity, but with larger mycobacterial loads the area of immunoperoxidase stain measured on a semi-automated image analyser correlated with the numbers of bacilli observed. The immunoperoxidase method will be useful in the evaluation of residual antigen in studying the pathogenesis of experimental murine tuberculosis. In human mycobacterial granulomata, this immunohistochemical technique should provide an alternative method of estimating the extent of bacillary load: this approach may also provide evidence of mycobacterial infection from residual antigen deposits in the tissue when whole bacilli have been successfully cleared.

Histometric studies on cellular infiltrates of tuberculin tests in patients with sarcoidosis.

The density and microanatomical location of CD4 and CD8 lymphocytes and of monocytes/macrophages at the site of a tuberculin test were measured in 13 patients with sarcoidosis, and the results were compared with those seen in a group of healthy controls. The cellular infiltrate was significantly reduced in the sarcoid subjects compared with the controls for all cell phenotypes studied; the ratio of CD4 positive:CD8 positive lymphocytes was significantly increased in the sarcoid group. Clinically negative reactions showed substantial numbers of infiltrating mononuclear cells, although not as great as in clinically apparent reactions. A clinically negative tuberculin reaction does not necessarily imply anergy to the test substance and should not be termed "negative".

Analysis of naturally occurring delayed-type hypersensitivity reactions in leprosy by in situ hybridization.

Analysis of tissue lesions of the major reactional states of leprosy was undertaken to study the immune mechanisms underlying regulation of cell-mediated immunity and delayed-type hypersensitivity (DTH) in man. In situ hybridization hybridization of reversal reaction biopsy specimens for INF-gamma mRNA expression revealed a 10-fold increase in specific mRNA-containing cells over that observed in unresponsive lepromatous patients. Expression of huHF serine esterase, a marker for T cytotoxic cells, were fourfold increased in reversal reaction and tuberculoid lesions above that detected in unresponsive lepromatous individuals. Immunohistology of reversal reactions confirmed a selective increase of Th and T cytotoxic cells in the cellular immune response. Of interest, the microanatomic location of these serine esterase mRNA-containing cells was identical to the distribution of CD4+ cells. Analysis of erythema nodosum leprosum (ENL) lesions revealed differences in the underlying immune processes in comparison with reversal reaction lesions. Although phenotypic Th cells predominated in ENL lesions, IFN-gamma and serine esterase gene expression were markedly reduced. We suggest that reversal reactions represent a hyperimmune DTH response characterized by a selective increase of CD4+ IFN-gamma producing cells and T cytotoxic cells, which result in the clearing of bacilli and concomitant tissue damage. In contrast, ENL reactions may be viewed as a transient diminution of Ts cells and activity leading to a partial and transient augmentation in cell-mediated immunity, perhaps sufficient to result in antibody and immune complex formation, but insufficient to clear bacilli from lesions.

Histometric studies on cellular infiltrates of tuberculin tests in patients with haemophilia.

The number of microanatomical location of CD4 and CD8 lymphocytes, of cells bearing receptors for I12 and transferrin, and of monocyte/macrophages in the dermis at the site of a tuberculin test were measured in 13 patients with haemophilia (10 seronegative and three seropositive for human immunodeficiency virus (HIV]. The overall density of lymphocytes in the perivascular and diffuse parts of the infiltrate was similar to that reported in other groups of subjects without evidence of immunosuppression. The CD4:CD8 ratio of the infiltrating lymphocytes throughout the section showed an inverse relation with clotting factor consumption. There was no significant change in the CD4:CD8 ratio in the diffuse infiltrate at various levels into the dermis in tuberculin reactions in patients with haemophilia, unlike healthy controls and other groups with no evidence of immunosuppression, who have previously been shown to have increasing CD4:CD8 ratio with increasing depth into the dermis. The number of cells bearing receptors for I12 and transferrin and of monocyte/macrophages was related to total lymphocyte density in the infiltrate. There was no evidence of serious impairment of the cell mediated response to a long term recall antigen, but the relatively low preponderance of CD4 lymphocytes in the diffuse infiltrate, particularly in the deeper dermis, may be the earliest indicator of impending immunosuppression.

Identification of binding sites for an insulin-like growth factor (IGF-I) in the median eminence of the rat brain by quantitative autoradiography.

The microanatomical location of IGF-I binding in the rat brain was determined by in vitro autoradiography with slide-mounted sections of frozen brain. Sections incubated in 0.1 nM [125I]-iodo-IGF-I produced a dense grain concentration in regions of the autoradiographic image corresponding to the external palisade zone of the median eminence; other hypothalamic regions were not so heavily labeled. This reaction was significantly reduced in the presence of 100 nM IGF-I. Measurement of binding by computer digital image analysis of autoradiographic images showed that specific binding for IGF-I in the median eminence was 41.3 +/- 8 X 10(-3) fmol/mm2 (mean +/- SEM); nonspecific binding was 11.9 +/- 1.8 X 10(-3) fmol/mm2. In contrast, specific binding to other hypothalamic regions was uniformly lower. In a separate experiment, 1000 nM unlabeled insulin was added. Without insulin, specific binding was 23 +/- 0.9 X 10(-3) fmol/mm2; nonspecific binding was 8 +/- 0.5 X 10(-3) fmol/mm2. In the presence of 1000 nM unlabeled insulin, specific binding for [125I]-iodo-IGF-I was 23 +/- 1 X 10(-3) fmol/mm2. The results suggest that a high concentration of receptors for an IGF-I-like molecule is present in the median eminence.

Histometric study of the localisation of lymphocyte subsets and accessory cells in human Mantoux reactions.

Intradermal injection of purified protein derivative produced typical delayed type hypersensitivity reactions in five healthy human subjects. The major subpopulations of lymphocytes and certain accessory cells were located in frozen sections of biopsies of the lesions with monoclonal antibodies and immunohistochemical staining. The densities (expressed as number/unit area for comparison) of the different types of cells were counted at various microanatomical locations in the tissue. The inflammatory cells were concentrated in narrow zones, initially (24 h) only surrounding small blood vessels but later (48-96 h) also around sweat ducts. Lymphocytes were the predominant cell type at these sites with T4 and T8 cells randomly intermixed at a ratio similar to that in the mononuclear cell fraction of the peripheral blood samples removed at the time of biopsy. There was also a scanty diffuse infiltrate in the intervening dermis, but here the T4:T8 ratio was significantly lower than that in the peripheral blood or perivascular cuffs. There was considerable intersubject variation in the relative preponderance of T8 cells in the diffuse infiltrate. The results suggest that there is no subset selection in the initial emigration of lymphocytes through vascular endothelium in the delayed hypersensitivity reaction, but that the subsets behave differently during the subsequent migration through the tissues. It remains to be determined whether the extent to which T8 cells migrate more rapidly than T4 cells through the tissues may influence the reaction at the site of entry of organisms or antigens into the body by altering the balance of the immunoregulatory lymphocyte subsets. This may underlie some of the differences in susceptibility to infection between subjects and determine the type of granuloma that develops in a particular patient.

Demonstration in situ of subsets of T-lymphocytes in sarcoidosis.

Five biopsy specimens of skin, four of lung, and one of a lymph node were taken from nine patients with sarcoidosis. Monoclonal antibodies were applied to frozen sections of the specimens by an immunoperoxidase technique to test for the presence and distribution of subsets of T-lymphocytes. T-cells expressing the suppressor/cytotoxic phenotype were found predominantly in lymphocytic mantles surrounding sarcoidal granulomas, whereas cells displaying the helper/inducer phenotype were distributed diffusely throughout granulomas. The ratio of helper to suppressor phenotypes in cutaneous sarcoidosis was 5.1 +/- 1.8. The microanatomic location of subpopulations of T-lymphocytes may be important in the pathogenesis of the granulomatous response of sarcoidosis.