In vitro biomineralization by osteoblast-like cells. II. Characterization of cellular culture supernatants

The quantification of total calcium, phosphorus, iron, chromium and nickel in cell culture medium by electrochemical or spectroscopic means may require digestion of samples. Nevertheless, when pH adjustment is performed for values higher than about 6.5, the formation of two phases occurs: a white precipitate and a clear solution. Analysing both phases using microelectrodes, atomic absorption spectrometry (AAS), diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, X-ray dispersive (XRD) analysis, scanning electron microscopy (SEM) and energy dispersive spectroscopic (EDS) analysis, it was observed that iron, chromium and nickel are not co-precipitating with the white solid phase. If quantification of calcium, phosphorus and magnesium is intended, a ten-fold dilution at least, must be performed to avoid most of these elements going into the precipitate. This knowledge is crucial if a mineralization study is going to be made.     

In vitro immune modulation by antibodies coupled to tumour cells

Modification of autologous tumour cells to express the immune costimulator B7.1 is a potential strategy for immunotherapy of cancer. Previously, this has involved introduction of genetic material into cells, in vitro culture, and confirmation of the protein product on the cell surface. This is possible only if sufficient tumour is obtainable and efficiently modified in a short time. Whilst progress has been made on ex vivo tumour cell culture and transfection/infection procedures there are still tumour types for which the present means of gene transfer are not efficient enough. We describe a highly efficient in vitro procedure for the modification of over 99% of the cells in a population, allowing the expression of cell surface proteins with potential immune modulatory activities. This procedure, which can be completed in as little as 24 h with no upper limit on cell number, utilizes succinimide esters to label cell surface proteins with biotin covalently. Biotinylated cell membrane proteins then anchor an avidin bridge for immobilizing protein G'-biotin. This can serve to bind immunoglobulin (Ig) molecules via their Fc region such that the variable region of the antibody is freely and functionally available. In the present study the binding of a stimulatory mouse anti-human CD28 monoclonal antibody to the surface of tumour cells is used to show that the modified cells are capable of co-stimulating T cells in vitro. The simplicity of the method, and the use of common reagents, represents a further step towards a realistic, truly 'off-the-shelf', nongene immunotherapy protocol.     

In vivo and in vitro macrophage activation by systemic adjuvants

Six systemic adjuvants of immunity were tested for their ability to induce macrophage activation. Four of them: living BCG, hydrosoluble extracts from BCG (HIU II) and from M.smegmatis (IPM), and lipopolysaccharide from E.coli (LPS), when administered to normal mice render macrophages non-specifically cytotoxic for tumor cells in vitro. The intensity of this phenomenon varied according to the route and time of adjuvant administration. In contrast, lentinan extracted from Lentinus edodes, and levamisole which is a synthetic chemical compound, depressed macrophage cytotoxic potential. BCG, IPM and LPS were shown to have a direct action on macrophages. After in vitro exposure to these agents, the cytotoxic potential of normal macrophages was greatly increased. Levamisole was unable to stimulate this macrophage function directly in vitro. On the other hand, such a macrophage activation has been induced in vitro when normal macrophages were cultivated in the presence of MIF coming from the supernatant of human lymphoblastoid cell lines.     

In vitro diagnosis of axillary lymph node metastases in breast cancer by spectrum analysis of radio frequency echo signals

Axillary lymph node status is of particular importance for staging and managing breast cancer. Currently, axillary lymph node dissection is performed routinely in cases of invasive breast cancer because of the lack of accurate noninvasive methods for diagnosing lymph node metastasis. We investigated the diagnostic ability of ultrasonic tissue characterization based on spectrum analysis of backscattered echo signals to detect axillary lymph node metastasis in breast cancer in vitro compared with in vitro B-mode imaging. Immediately after surgery, individual lymph nodes were isolated from axillary tissue. Each lymph node was scanned in a water bath using a 10-MHz instrument, and radio frequency data and B-mode images were acquired. Spectral parameter values were calculated, and discriminant analysis was performed to classify metastatic and nonmetastatic lymph nodes. Forty histologically characterized axillary lymph nodes were enrolled in this study, including 25 nonmetastatic and 15 metastatic lymph nodes. A significant difference existed in the spectral parameter values (slope and intercept) for metastatic and nonmetastatic lymph nodes. Spectral parameter-based discriminant function classification of metastatic vs. nonmetastatic lymph nodes provided a sensitivity of 93.3%, specificity of 92.0%, and overall accuracy of 92.5%. In comparison, B-mode ultrasound images of in vitro lymph nodes provided a sensitivity of 73.3%, specificity of 84.0%, and overall accuracy of 80.0%. Receiver operating characteristic (ROC) analysis comparing the efficacy of both methods gave an ROC curve area of 0.9888 for spectral methods, which was greater than the area of 0.8980 for B-mode ultrasound. Hence, this in vitro study suggests that the diagnostic ability of spectrum analysis may prove to be markedly superior to that of B-mode ultrasound in detecting axillary lymph node metastasis in breast cancer. Because of these encouraging results, we intend to conduct an investigation of the ability of spectral methods to classify metastatic axillary lymph nodes in vivo.     

In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1

Cellular pathways for induction of programmed cell death (PCD) have been identified, but little is known about specific extracellular matrix processes that may affect apoptosis along those pathways. In this study, a series of Burkitt's lymphoma (BL) cell lines were assayed for their expression of tissue inhibitor of metalloproteinases (TIMP)-1. Results indicate that TIMP-1-positive BL lines show resistance to cold-shock-induced apoptosis. Furthermore, recombinant TIMP-1, but not TIMP-2 or a synthetic metalloproteinase inhibitor (BB-94), confers resistance to apoptosis induced by both CD95-dependent and -independent (cold shock, serum deprivation, and gamma-radiation) pathways in TIMP-1-negative BL lines. TIMP-1 suppression of PCD is not due to metalloproteinase inhibition, as reduction and alkylation of the TIMP-1 did not abolish this activity. Retroviral induction of TIMP-1 not only resulted in cell survival but also in continued DNA synthesis for up to 5 d in the absence of serum, while controls underwent apoptosis. This resistance to apoptosis is reversed by anti-TIMP-1 antibodies, demonstrating that secreted TIMP-1 is active in blocking apoptosis. Furthermore, TIMP-1 upregulation induced expression of Bcl-XL but not Bcl-2 as well as decreased NF-kappaB activity as compared with controls. These results demonstrate that TIMP-1 suppresses apoptosis in B cells and suggests a novel activity for TIMP-1 in tissue homeostasis.     

Adhesion molecule expression by epidermal Langerhans cells and lymph node dendritic cells: a comparison

BACKGROUND: Nasal gliomas are uncommon neurogenic malformations, which derive from the prenasal space. They may appear as intranasal masses, frontonasal masses, or deformities of the nose, brow, or lower central forehead. Almost all of these tumors were diagnosed shortly after birth. The clinical findings of meningoencephaloceles, nasal fistulas, dermoides and epidermoid cysts are presented additionally for differential diagnosis. PATIENTS: Following some interesting case reports, the management of these types of benign tumors is discussed. RESULTS: Complete radiologic evaluation with computed tomography and magnetic resonance imaging should be performed, because a possible intracranial connection must be considered. The preferred surgical treatment of glioma is an endoscopically controlled procedure. In most cases craniotomy is not required. Open rhinoplasty can be helpful for removal of ectodermic malformations. CONCLUSIONS: The surgeon should be familiar with the diagnosis and management of the rare congenital tumors of the nose to ensure proper therapy and to provide the requisite information for patients, parents, and colleagues.     

Lymphocytocidal lymphocyte trapping by human lymph node cells: a tissue culture-ultrastructural study

An ultrastructural study was carried out on 25 lymphocyte-trapping cells selected from tissue cultures of human axillary lymph nodes. The trapping cells contained several hundred intravacuolar lymphocytes, most of which showed degenerative changes. The principal findings are: (a) a braod spectrum of lymphocyte degeneration; (b) a consistent pattern of lymphocyte degeneration beginning with perinuclear vacuoles and ending with breakdown of the nuclear envelope; (c) the viable lymphocytes tended to be located in a juxtanuclear region; (d) a lysosomal relationship was suggested for lymphocyte degeneration but not for lymphocyte trapping; and (e) degeneration of the trapping cell, or lymphocytes associated with other cells, was not observed. The sequence of degenerative changes differs from those reported for several classes of lymphocytocidal agents. There were no morphologic properties of the trapping cell which served to identify it more specifically. The findings, together with previous time-lapse film observations, warrant further investigation of the hypothesis that lymphocytocidal lymphocyte trapping may be involved in the control of lymphocyte populations.     

In vitro modulatory effects of interleukin-3 on macrophage activation induced by interleukin-2

BACKGROUND: The concomitant activation of macrophage-mediated immunosuppressive events might represent one of the most important biologic factors responsible for the decreased efficacy of cancer immunotherapies, including that of interleukin (IL)-2. In previous studies, the authors observed that the increase in the soluble IL-2 receptor (SIL-2R) and neopterin levels was related to the generation of macrophage-mediated immunosuppression and associated with a reduced clinical efficacy during IL-2 cancer immunotherapy. Because both cytokines and neurohormones may influence the macrophage system, the current study was done to evaluate the effects of IL-3 and of the pineal hormone melatonin (MLT) on monocyte response to IL-2 administration. METHODS: Peripheral blood monocytes were incubated with different concentrations of IL-2, IL-3, and MLT, either alone or in association. RESULTS: SIL-2R, neopterin, and tumor necrosis factor-alpha mean concentrations in the medium significantly increased during incubation with IL-2 at a concentration of 100 Cetus units/ml. IL-3 alone, at a dose of 10 ng/ml, also stimulated tumor necrosis factor release; no effect was found on SIL-2R and neopterin. The IL-2-induced neopterin rise was blocked by a concomitant incubation with IL-3 at a dose of 10 ng/ml. Finally, the concomitant incubation with IL-3 and MLT further inhibited neopterin release and significantly decreased IL-2-induced SIL-2R secretion. CONCLUSIONS: These results suggest that IL-3 alone or in association with MLT may modulate macrophage functions during cancer immunotherapy with IL-2 and decrease the IL-2-induced macrophage activation.     

Predicting regional lymph node metastasis in carcinoma of the penis: a comparison between fine-needle aspiration cytology, sentinel lymph node biopsy and medial inguinal lymph node biopsy

OBJECTIVE: To evaluate the accuracy of clinical examination and fine-needle aspiration cytology (FNAC) in detecting groin metastases in patients with carcinoma of the penis, and to assess the positive and negative predictive value (PPV, NPV) of a preliminary sentinel lymph-node biopsy (SNB) and biopsy of the most medial of the horizontal group of inguinal lymph nodes (MIN) in selecting patients for an ilio-inguinal block dissection. PATIENTS AND METHODS: The study comprised 28 patients (56 groins) with Stage I (one), Stage II (11) and Stage III (16) carcinoma of the penis. All patients underwent a detailed clinical examination followed by FNAC of the palpable inguinal nodes, and were subsequently submitted for block dissection. The MIN, the SN and the rest of the inguinal and iliac nodes were histologically examined separately for metastases. RESULTS: The clinical evaluation had a sensitivity of 74%, a specificity of 61%, a PPV of 57% and a NPV of 77%. The corresponding values for FNAC were all 100%, and the specificity and PPV for both MIN and SN were 100%. The sensitivity and NPV of MIN were higher than for SN, although not significantly so. CONCLUSION: Clinical examination alone is inaccurate in selecting patients with carcinoma of the penis for block dissection. FNAC is accurate and specific when nodes are palpable; in those with impalpable nodes a preliminary MIN biopsy followed by SNB if the MIN biopsy is negative will accurately select all patients with metastases in the groin nodes. This can be performed by examining frozen sections of the lymph nodes; if positive, block dissection can be carried out at the same time.     

Differential regulation of tissue-specific lymph node high endothelial venule cell adhesion molecules by tumour necrosis factor and transforming growth factor-beta 1

Lymphocytes migrate from blood into lymph nodes (LN) of rats specifically at segments of venules lined by high endothelium (HEV). We have previously shown that pretreatment of LN HEV cells with pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), augments their adhesiveness for thoracic duct lymphocytes (TDL). Here we report that a mouse monoclonal antibody, 3C10, recognized tissue-specific endothelial determinants on rat LN HEV cells and blocked their adhesiveness for TDL and EL-4J cells transfected with rat L-selectin. In contrast, 3C10 antibody did not inhibit lymphocyte attachment to Peyer's patch (PP) frozen sections or cultured PP HEV cells. The antibody immunoprecipitated from LN HEV cells two proteins with apparent molecular weights of 90,000 and 50,000. The expression of 3C10 antigen on LN HEV cells was increased by incubation with TNF-alpha or IFN-gamma. Furthermore, pretreatment of cytokine-stimulated LN HEV cells with 3C10 antibody blocked TDL binding in a dose-dependent manner. In contrast, 3C10 antigen expression on LN HEV cells was significantly decreased following incubation of cells with transforming growth factor-beta 1 (TGF-beta 1). In addition, TGF-beta 1 also abrogated the adhesiveness of LN HEV cells stimulated with TNF-alpha, IFN-gamma or both cytokines. Together, these data suggest that endothelial determinants recognized by the 3C10 antibody are tissue-specific ligands for lymphocyte adhesion and cytokines such as TNF-alpha and TGF-beta differentially regulate their expression and function.     

Heterogeneity of dendritic cells in human superficial lymph node: in vitro maturation of immature dendritic cells into mature or activated interdigitating reticulum cells

A target identification paradigm was used to study cross-modal spatial cuing effects on auditory and visual target identification. Each trial consisted of an auditory or visual spatial cue followed by an auditory or visual target. The cue and target could be either of the same modality (within-modality conditions) or of different modalities (between-modalities conditions). In 3 experiments, a larger cue validity effect was apparent on within-modality trials than on between-modalities trials. In addition, the likelihood of identifying a significant cross-modal cuing effect was observed to depend on the predictability of the cue-target relation. These effects are interpreted as evidence (a) of separate auditory and visual spatial attention mechanisms and (b) that target identification may be influenced by spatial cues of another modality but that this effect is primarily dependent on the engagement of endogenous attentional mechanisms.     

Antigen-specific IgA response of NALT and cervical lymph node cells in antigen-primed rats

Three models to estimate energy requirement as a function of growth curve pattern were applied to controlled experimental data of male vs female of broilers and turkeys. The share of maintenance out of total feed requirement was 55% for the average of the three models with major divergence due to age. Comparison of the ratio between actual and estimated feed consumption reveals that the relative energy requirement was always lower in females than in males in the range of 5 to 10% for the three models, with an average of 7.7%. It appears, therefore, that in estimating the energy requirement for use in practical feeding, specific models should be assigned for males and females in both broilers and turkeys.     

In vitro production of plasminogen activator by human granulosa cells

Plasminogen activator (PA) production by granulosa cells has been demonstrated in several species. In the human ovary, tissue-type PA and urokinase-type PA antigens have been found in the follicular fluids, but neither PA activity nor mRNA for both enzymes was found in granulosa cells of preovulatory follicles. All of these studies were performed on granulosa cells collected from follicles immediately before ovulation, when the cells were already in the luteal phase. In the attempt to better characterize the PA/plasminogen system in the human ovary, we examined PA and PA inhibitor (PAI) production in cultures of granulosa cells obtained from normally cycling untreated women at different stages of the cycle. In addition, we analyzed granulosa-luteal cells obtained from hormonally stimulated women undergoing gamete intrafallopian tube transfer, as a model of late phase follicular development. Zymographic analysis as well as immunoprecipitation with specific antisera revealed that granulosa cells from follicles at early phases of antral stages secreted high levels of PA of the urokinase type in the medium. No free tissue-type PA activity was found in any of the examined samples. On the contrary, free PAI was undetectable in medium obtained from granulosa cell cultures, and it was abundant in granulosa-luteal cell cultures, where it was found in two forms. These data show that in the human ovary as in that of the rat, PAs and PAIs are tightly time regulated. The timing of PA production in human granulosa cells suggests a role for PA activity at early stages of follicular maturation.     

In vitro infection of smooth muscle cells by Chlamydia pneumoniae

Recent observations have shown that both Chlamydia pneumoniae antigens and DNA may be found within atherosclerotic lesions. In this study, we evaluated the ability of C. pneumoniae to infect cells that make up atherosclerotic lesions, including endothelial cells, smooth muscle cells, and cholesterol-loaded smooth muscle cells. The organism readily infected rabbit, bovine, and human aortic smooth muscle cells. Cholesterol-loaded smooth muscle cells were even more susceptible to C. pneumoniae infection. Chlamydia trachomatis inefficiently infected smooth muscle cells, demonstrating that this is not a characteristic of all members of the genus Chlamydia. C. pneumoniae infected bovine endothelial cells poorly. This study demonstrates that C. pneumoniae readily infects one of the important types of cells found within atherosclerotic lesions, i.e., smooth muscle cells with and without cholesterol loading.