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Aims Adenomyoepithelioma (AME) and adenoid cystic carcinoma (ACC) of the breast have been noted to occur simultaneously, raising the possibility that AME may represent a related or precursor lesion to ACC. ACC frequently harbours genetic rearrangement of the MYB gene. We sought to clarify the relationship between AME and ACC by comparing their rates of MYB expression by IHC and MYB rearrangement by FISH. Methods and results IHC and FISH for MYB rearrangement were performed on paraffin-embedded sections of 11 breast ACCs, 11 non-breast ACCs and 11 breast-AMEs. Using FISH, five of eight (63%) interpretable breast ACCs demonstrated MYB gene rearrangement. Nine of 11 (81%) breast ACCs demonstrated MYB expression (range = 20-95%). Of the three FISH-negative breast ACCs, two were solid variant and demonstrated strong MYB expression by IHC. Of the 10 interpretable non-breast ACCs, six showed MYB rearrangement, all of which were conventional type. Nine of these 11 (81%) cases showed MYB immunoexpression (range = 10-90%), including three solid-variant cases which were negative by FISH. No MYB rearrangements were detected by FISH in 10 interpretable AMEs. However, three of 11 cases (27%) showed weak to moderate MYB expression by IHC (range = 10-40%). Conclusions Our results indicate that AMEs do not harbour MYB gene rearrangement. IHC for MYB may be helpful in diagnosing FISH-negative cases of ACC, particularly the diagnostically more difficult solid variants. However, weak to moderate MYB expression in a subset of AMEs highlights not only a potential diagnostic pitfall, but also shared pathophysiology with ACC worth investigating further at the genomic level.

Olfactory neuroblastoma and the management of neck disease has posed considerable challenges to the treating physician. The aims of the study were to determine the incidence and factors influencing neck disease and to identify at-risk patients with cervical node-negative disease at presentation.Multicenter case series with retrospective chart review.In sum, 113 patients with a histopathologic diagnosis of olfactory neuroblastoma across 6 tertiary hospitals in Australia and the United States.Treatment modalities for the primary site and neck included surgery, radiotherapy, and combined therapy. Treatment outcomes were measured in relation to date of primary treatment, and long-term follow-up was recorded. Disease-free survival was calculated as time for patients to develop delayed neck disease following primary treatment.A total of 113 patients (46 females, 49.7 ± 13.2 years) were identified with a median follow-up of 41.5 months (interquartile range, 58.2); 7.1% of patients presented with primary neck disease, while 8.8% of patients presented with delayed neck disease. Neck disease was present in patients with Hyams grade II (22.2%), III (55.6%), and IV (22.2%) lesions (χ(2) = 5.66, P = .13). Histologic grade was higher in patients with primary neck disease (χ(2) = 16.22, P = .001). Positive surgical margins were associated with a higher risk of delayed neck disease as compared with clear surgical margin (17.9% vs 5%, P = .034).Neck metastasis is an important clinical consideration for olfactory neuroblastoma at presentation and in surveillance. Primary treatment of the neck could be considered in select patients. Long-term surveillance of the neck and primary site is essential.

Background: To identify computer extracted in vivo dynamic contrast enhanced (DCE) MRI markers associated with quantitative histomorphometric (QH) characteristics of microvessels and Gleason scores (GS) in prostate cancer. Methods: This study considered retrospective data from 23 biopsy confirmed prostate cancer patients who underwent 3 Tesla multiparametric MRI before radical prostatectomy (RP). Representative slices from RP specimens were stained with vascular marker CD31. Tumor extent was mapped from RP sections onto DCE MRI using nonlinear registration methods. Seventy-seven microvessel QH features and 18 DCE MRI kinetic features were extracted and evaluated for their ability to distinguish low from intermediate and high GS. The effect of temporal sampling on kinetic features was assessed and correlations between those robust to temporal resolution and microvessel features discriminative of GS were examined. Results: A total of 12 microvessel architectural features were discriminative of low and intermediate/high grade tumors with area under the receiver operating characteristic curve (AUC) > 0.7. These features were most highly correlated with mean washout gradient (WG) (max rho 52 0.62). Independent analysis revealed WG to be moderately robust to temporal resolution (intraclass correlation coefficient [ICC] 5 0.63) and WG variance, which was poorly correlated with microvessel features, to be predictive of low grade tumors (AUC 5 0.77). Enhancement ratio was the most robust (ICC 5 0.96) and discriminative (AUC 5 0.78) kinetic feature but was moderately correlated with microvessel features (max rho 52 0.52). Conclusion: Computer extracted features of prostate DCE MRI appear to be correlated with microvessel architecture and may be discriminative of low versus intermediate and high GS. J. MAGN. RESON. IMAGING 2015;00:000–000.

Aim: Caesarean section (CS) deliveries have increased, mostly because of patient/obstetrician preference. Although CS decreases the risk of delivery-related injuries, it increases the risk for respiratory and neurological complications. Complication rates are reportedly higher for elective CSs for term infants performed at 37–38 gestational weeks than later. We investigated this difference in an Israeli cohort. Methods: Data on all births in our medical centre during 2007–2009 were reviewed. Those on elective CSs for term infants were retrieved and divided into ‘early’ (37–38 gestational weeks) or ‘late’ (week 39 or later) groups whose epidemiological and outcome characteristics were compared. Result: Of the 12 276 births, 596 were early and 454 were late elective CSs. There were no differences in gender, ethnicity, Apgar score or length of hospital stay. Twenty-six infants from the early group and 11 infants from the late group were transferred to the neonatal intensive care unit. Within them, compared with all elective CSs, the morbidity rate was higher for the former infants than for the latter. Conclusion: Morbidity was higher among infants who were delivered at 37–38 gestational weeks by elective CS. We recommend postponing elective CSs to ≥39 weeks.

Single-molecule fluorescence imaging has provided unprecedented access to the dynamics of ribosome function, revealing transient intermediate states that are critical to ribosome activity. Imaging platforms have now been developed that are capable of probing many hundreds of molecules simultaneously at temporal and spatial resolutions approaching the sub-millisecond time and the sub-nanometer scales. These advances enable both steady- and pre-steady state measurements of individual steps in the translation process as well as processive reactions. The data generated using these methods have yielded new, quantitative structural and kinetic insights into ribosomal activity. They have also shed light on the mechanisms of antibiotic targeting the translation apparatus, revealing features of the structure-function relationship that would be difficult to obtain by other means. This review provides an overview of the types of information that can be obtained using such imaging platforms and a blueprint for using the technique to assess how small-molecule antibiotics alter macromolecular functions.

Aims To investigate the use of a computer-assisted technology for objective, cell-based quantification of molecular biomarkers in specified cell types in histopathology specimens, with the aim of advancing current visual estimation or pixel-level (rather than cell-based) quantification methods.

Objectives/Hypothesis: Our goal was to develop a noninvasive, dynamic imaging method that would further the understanding of head and neck cancer (HNC) tumor growth and local spreading. We developed a novel orthotopic mouse model of HNC with a stable cell line expressing a red fluorescent protein gene to compare a molecular imaging tumor quantification with traditional caliper measurement. Methods: An HNC-tdT stable cell line expressing the tdTomato gene was established, which were injected into the floor of the mouth of nude mice. Tumor growth was constantly monitored using molecular imaging for up to 35 days. The tumors were further evaluated by histologic examination. Results: Established tumors consistently expressed fluorescent signals that were successfully imaged by molecular imaging during the study. Initial tumor development was detected earlier than caliper measurement would allow. The fluorescent signal quantities of tumors detected by the imaging correlated with the tumor sizes measured by calipers. Conclusions: This novel animal model represents an orthotopic human HNC model. The tumor can be detected earlier with molecular imaging than by conventional external caliper measurement. Unlike surgical measurement, the tumor can be quantified without disturbing the tumor environment. This model has significant potential for HNC oncologic research.

Nanotubes have great potential for applications in a rapidly increasing range of fields: catalysis, medicine, pharmacy, pheromone-release systems for crop protection, sensorics, and photonics. This is due to their high anisotropy, huge specific surface area that enhances reactivity, high rate of adsorption, and efficiency of transport processes both within and across the nanotube walls. Electrospinning is a process that produces continuous polymer fibers through the action of an external electric field imposed on a polymer solution (for a review of this process see previous publications). To manufacture nanotubes, two fundamentally different approaches have been reported: self-assembly and template-based methods. In the TUFT (tubes by fiber templates) process, electrospun nanofibers themselves are used as templates to produce nanotubes. The template-based TUFT process for nanotube production consists of three stages: i) the electrospinning of template nanofibers, ii) shell deposition via chemical or physical vapor deposition (CVD or PVD, respectively), and iii) core removal by thermal or chemical means. Recently, a new technique was introduced that allows the co-electrospinning of polymer solutions from a spinneret containing two coaxial capillaries. Using this technique, coelectrospinning of immiscible and miscible pairs of polymer solutions produced nanofibers with core/shell structures. This technique was then used to co-electrospin conjugated polymer nanofibers and to make the PCL/gelatin (PCL: poly(e-caprolactone)) core/shell structure that holds great potential for controlled drug delivery and as a scaffold for tissue engineering. It was possible to co-electrospin almost non-spinnable polymers such as the conducting polymer polyaniline (PAni). Using this same co-electrospinning technique, ceramic sol–gel precursors were added to the shell solutions to create ceramic nanotubes. The core material—a heavy mineral oil—was later extracted with octane. The aim of the present work is to produce hollow carbon nanotubes by co-electrospinning two polymer solutions. The process was carried out in two stages. In the first stage, use was made of the non-solvent effect on one of the polymers to facilitate the creation of a solid interface between the nanofiber’s core and shell. In the second stage, the nanofibers were subjected to heat treatment to degrade the core polymer and carbonize the polymer shell. Figure 1 shows a typical pattern of the co-electrospinning process close to the core/shell spinneret. The core-polymer capillary protrudes 0.3 to 1 mm below the shell capillary. As

Objective: This study examines preoperative clinical and intraoperative histopathologic characteristics that can be used to predict thyroid gland invasion in the setting of squamous cell carcinoma (SCC) of the glottis. Study Design: The study was retrospectively performed using 30 serially sectioned whole-organ total laryngectomy with thyroidectomy specimens with associated preoperative clinical data. Methods: Histopathologic and clinical variables including true vocal cord (TVC) fixation, cricoarytenoid joint invasion, subglottic extension (SGE) of tumor, patterns of laryngeal spread, and prior radiation were examined as univariate and multivariate correlates of thyroid gland invasion. Results: Twenty-three percent of thyroid gland specimens demonstrated SCC invasion. Five were T4 stage, two were T3 stage, and all demonstrated direct extension to the thyroid gland. Of these, all had a fixed ipsilateral TVC (P = .003) and SGE of tumor greater than 15 mm (P = .003). Using multivariate analysis, SGE of tumor and TVC fixation contribute independently as correlates of thyroid gland invasion. Prior radiation of the larynx did not correlate with thyroid gland invasion and did not significantly influence the predictive capacity of these variables. Tumors invading the thyroid gland also invaded the cricothyroid membrane (100%), anterior commissure (100%), laryngeal ventricle (100%), and thyroid cartilage (86%). Conclusion: Preoperative assessment of TVC mobility and extent of SGE are significant correlates of thyroid gland invasion by SCC of the glottis. Distinct patterns of laryngeal spread are associated with thyroid gland invasion. Prophylactic hemithyroidectomy with isthmusectomy is indicated for glottic SCC in the preoperative setting of a fixed TVC and SGE greater than 15 mm. Additional study correlating patterns of laryngeal spread with thyroid gland invasion will add to these data in determining when to selectively perform thyroidectomy in this setting.

Objectives/Hypothesis: The study examined preoperative clinical characteristics that can be used to predict secure inferior margins of glottic squamous cell carcinoma extending toward the cricoid cartilage when performing organ preservation surgery of the larynx. Study Design: The study was retrospectively performed using 31 serially sectioned whole-organ total laryngectomy specimens with associated preoperative clinical data. Methods: Histopathologic and clinical variables including true vocal cord (TVC) fixation, cricoarytenoid joint invasion, subglottic extension (SGE) of tumor, and prior radiation were examined as independent and multivariate correlates of cricoid cartilage invasion. Results: All tumors with subglottic extension of 15 mm or less and without arytenoid fixation were free of cricoid invasion. Of tumors invading cricoid with subglottic extension of 15 mm or less, all had a fixed arytenoid cartilage and local cricoid invasion type only. Correcting for subglottic extension using multivariate analysis, cricoarytenoid joint invasion and fixed true vocal cord independently predicted cricoid invasion. However, in a multivariate model together, true vocal cord mobility adds no predictive power to cricoarytenoid joint invasion. Prior radiation of the larynx did not significantly change the predictive capacity of these variables. Conclusion: Preoperative assessment of arytenoid mobility and extent of subglottic extension are reliable predictors of cricoid invasion by glottic squamous cell carcinoma. Organ preservation surgery is oncologically safe in the setting of glottic squamous cell carcinoma with subglottic extension of 15 mm or less and without arytenoid fixation.

Objectives/Hypothesis Perineural invasion (PNI) is increasingly being recognized as an important indicator of aggressiveness in head and neck squamous cell carcinoma. The mechanisms of PNI are poorly understood. Laminin-5, an important basement membrane constituent, has been shown to be essential in head and neck squamous cell carcinoma invasion and motility. We hypothesized that tumors exhibiting increased expression of laminin-5 are more likely to be neurotropic. Study Design Analysis of archived surgical specimens with and without PNI for presence and intensity of laminin-5 tumor staining. Methods Immunohistochemistry of archived head and neck squamous cell carcinoma specimens with known PNI was performed with anti–laminin-5 antibodies and appropriate positive and negative control specimens. The staining patterns were characterized as follows: A, few to no tumor cells positive; B, some peripheral cells positive; C, all peripheral cells positive; and D, almost all tumor cells positive. Statistical analysis was by χ2 analysis. Results Forty-six PNI-positive and 18 PNI-negative specimens were analyzed. The staining distribution for the PNI-positive specimens was as follows: 2% for A, 41% for B, 46% for C, and 11% for D. For tumors without PNI, the distribution was 28% for A, 50% for B, 22% for C, and 0% for D (P = .005). In PNI-positive tumors, no significant difference in staining was seen between areas with and without PNI. Conclusions We found a significant correlation between laminin-5 staining and the presence of PNI in head and neck squamous cell carcinoma. Expression of laminin-5 by tumors is, possibly, an important step in the process of PNI. These preliminary findings support the concept that deposition of basement membrane constituents are required in the multistep process of nerve invasion.

The MUC1 protein was found to be up-regulated in a spectrum of malignant tumors. T-cell responses to the MUC1 extracellular tandem repeat array (TRA) were observed in murine models as well as in breast-carcinoma patients. In the present study, we evaluated the anti-tumor potential of HLA-A2.1-motif-selected peptides from non-TRA domains of the molecule. Peptide immunogenicity was examined in the Db-/- x beta2 microglobulin (beta2m) null mice transgenic for a modified HLA-A2.1/Db-beta2 microglobulin single chain (HHD mice). Our results show the existence of 3 novel HLA-A2.1-restricted MUC1-derived cytotoxic T-lymphocyte (CTL) epitopes. These peptides are processed and presented by the HHD-transfected breast-tumor cell line MDA-MB-157. Moreover, CTL induced by these 3 peptides show higher lysis of target cells pulsed with breast-carcinoma-derived peptides than of targets pulsed with normal breast-tissue-derived peptides. These data suggest an important role for non-TRA MUC1-derived peptides as inducers of a MHC-restricted CTL reaction to a breast-carcinoma cell line and patient-derived tumor extracts.

Epitope spreading is a process whereby epitopes distinct from and non-cross-reactive with an inducing epitope become targets of an evolving immune response. This phenomenon has been associated most notably with the progression of naturally occurring or experimentally induced chronic autoimmune diseases. We have investigated the potential occurrence of epitope spreading in the context of antitumor cytotoxic T cell (CTL) responses using chicken ovalbumin (OVA) as a model antigen. Our results indicate that following rejection of OVA-expressing EG.7 tumor cells effectuated by a CTL response which is induced against the MHC class I-restricted immunodominant epitope OVA257-264, there occurs intramolecular diversification of the CTL response to two additional OVA-derived epitopes, OVA176-183 and OVA55-62, as well as intermolecular spreading to other endogenous tumor-derived determinants. It seems that CTL-mediated tumor cell destruction in vivo favors cross-presentation of additional epitopes with the consequent activation of additional tumor-reactive lymphocytes. The process of epitope spreading in that context has obvious important implications for the design of antigen-specific antitumor immunotherapies.

We have partially characterized the T-cell subsets that control the growth of the C57BL/6 Lewis lung carcinoma 3LL transplanted into syngeneic mice. By analyzing the phenotypes of anti-tumor lymphocytes generated in vitro and in vivo, we have characterized a CD8 T-cell receptor (TcR) V beta 5,6 positive subpopulation of cytotoxic effectors important in retarding the growth of the transplanted tumor. In contrast, the rejection of 3LL appears to be hindered by the presence of a CD4 V beta II-positive subset since depletion of these lymphocytes in vivo with the appropriate monoclonal antibodies (MAbs) results in significant retardation of tumor growth. These results suggest that the cumulative positive and negative effects of distinct T-cell sub-populations determine the outcome of tumor progression and metastasis.

B16-F10.9 is a highly metastatic clone of the B16-F10 melanoma line, that expresses low levels of MHC class-I antigens. F10.9 cells transfected with H-2Kb are highly immunogenic and consequently exhibit a low metastatic phenotype. Treatment with gamma-IFN elevated H-2Kb and H-2Db cell surface expression of F10.9 cells to levels much higher than did transfection of these genes. Yet, following intravenous injection, the gamma-IFN treated cells generated high loads of lung metastases. However, when tested for their immunogenic effect, they elicited CTL and were sensitive to CTL. Immunization with both the positive transfectant KI and the gamma-IFN-treated F10.9 cells protected in vivo against metastatic spread of a subsequent transplant of parental F10.9 cells. The protection elicited by KI transfectants was more effective than the protection by gamma-IFN-treated cells.

The application of the Hilbert transform to the signal analysis provides some additional information about amplitude, instantaneous phase, and frequency of vibrations. The Hilbert transform decomposition methods are intended for extracting simple components using the varying instantaneous frequency and amplitude from multicomponent nonstationary signals. The corresponding identification methods directly extract the backbone and damping curve of nonlinear vibration systems. This enables us to solve an inverse identification problem, namely, the problem of estimation of the initial nonlinear static elastic and damping force characteristics. Keywords: Hilbert transform; analytic signal; envelope; instantaneous phase; instantaneous frequency; empirical mode decomposition; Hilbert vibration decomposition; nonlinear system identification

Problem: This study examines preoperative clinical characteristics that can be used to predict secure inferior margins of glottic squamous cell carcinoma (SCC) extending toward the cricoid cartilage, when performing organ preservation surgery of the larynx. Methods: Thirty-one serially sectioned whole-organ laryngectomies were histologically analyzed and true vocal cord (TVC) mobility, cricoarytenoid joint invasion, subglottic extension (SGE) of tumor, and prior radiation were examined as independent and multivariate correlates of cricoid invasion. Results: All tumors with SGE. Conclusion: Preoperative assessment of arytenoid mobility and extent of SGE are reliable predictors of cricoid invasion by glottic SCC. Significance: Organ preservation surgery is oncologically safe in the setting of glottic SCC with SGE. Support: None reported.

Background. Several studies have documented cutaneous findings in neonates of various racial groups. Our purpose was to determine the frequency of birthmarks in Israeli neonates of Jewish and Arabic origin. Methods. A cohort of 1672 newborn infants under 96 hours of age were examined for the presence of birthmarks. Of these 841 (50·3%) were Jewish and 831 (49·7%) were Arab. The Jewish group was further subdivided into various ethnic groups according to parental ancestry. Results. Melanocytic brown lesions (Mongolian spots, congenital nevi, and cafe-au-lait spots), were more common in Arab infants. The vast majority of Jewish infants with Mongolian spots were of Asian or African ancestry. On the other hand, congenital melanocytic nevi were found only in Jewish infants of European ancestry. Vascular lesions (salmon patch and port-wine stain) in Arab neonates exhibited a female preponderance. Conclusions. Our data suggest that the prevalence of birthmarks in Israeli neonates is similar to the prevalence reported by others in white neonates.

Quantified Immunohistochemistry Demonstrates Cellular Level Targets of an Oral Multikinase Inhibitor in Metastatic Thyroid Cancer Evan R. Ransom, MD1; Jason M. Leibowitz, MD1; Cathy Ma, MD2; Kanchan Puttaswamy, MS2; Andrea Troxel, ScD3; William Lee, PhD1; Michael Feldman, MD, PhD4; Marcia S. Brose, MD, PhD1,2 Department of 1Otolaryngology-Head&Neck Surgery, 2Hematology/Oncology, 3Biostatistics, & 4Pathology, University of Pennsylvania

Two clones of the 3LL Lewis lung carcinoma, a low-metastatic clone A9 and a high-metastatic clone D122, were studied for MHC expression and immunogenic properties. Using monoclonal antibodies, we demonstrated that the A9 clone expresses both the H-2Kb and the H-2Db, whereas the D122 expresses only the H-2Db, and lacks the expression of the H-2Kb encoded molecules. Cells of the low-metastatic clone A9 grew progressively in syngeneic (C57BL/6J) or in F1 mice, but were rejected in allogeneic recipients. The high-metastatic D122 grew progressively in all mouse strains tested, yet metastases were formed only in syngeneic recipients. When H-2 recombinant mice were used, the A9 again manifested a significantly greater immunogenic potency than the metastatic D122, which grew in all 4 recombinants tested. Metastases, however, were formed in B10HTG and to a lesser extent in B10A(4R), thus indicating that metastasis formation is restricted by both C57BL background and H-2Db sub region. We subsequently tested whether the higher immunogenicity of the H-2Kb-positive A9 cells is expressed also in syngeneic mice, to examine whether this could account for its low metastatic phenotype. We found that immunization by A9 cells significantly inhibited the growth of a subsequent A9 graft and even of D122, yet D122 did not retard the growth of secondary D122 or A9 cells. The increased immunogenic effect was expressed also in the generation of syngeneic cytotoxic lymphocytes by A9 but not by D122 cells. We suggest that expression of H-2K molecules on the 3LL clones, immunogenicity and the metastatic phenotype are causally related in this system.

Human normal T cells were selected for in vittro cloning according to the expression of T4, T8 or T10 antigens on individual cells. Clones were produced from each of these cells irrespective of the antigenic phenotype of the parental cell. The cloned progeny manifested, in many cases, shifts in antigen expression. Thus, T4+T8− cells gave clones expressing predominantly T4−T8+ and vice versa. The clonal expression of T4 and T8 seemed to be mutually exclusive. Antigenic shifts were recorded also in clones derived from T4−T8−T10− cells, resulting in T10+ clones which were also either T4+ or T8+ and from T4+T8−T10+ cloned cells yielding clones of either T4+ or T8+ cells. Testing functional properties we found that NK activity was mediated not only by T10+ cells but also, in some cases, by T4+ and T8+ cells. Moreover, TCGF production, which may reflect helper activity, was mediated not only by T4+ cells. Only the cytotoxic (CTL) activity seems to be confined to the T8 phenotype. Thus, it appears that T antigens, which seemed to be molecular markers of differentiation, are not markers for terminal differentiation and do not always reflect defined functional properties. These conclusions are drawn from cloning of normal T cells which manifest properties different from those of T-cell lines or T hybridomas.

The development of T lymphocyte immunocompetence appears to depend upon the function of the thymus gland. To study this function in greater detail, we developed a method for preparing monolayer cultures of thymus reticulum cells. We found that populations of lymphocytes from thymus-deprived mice acquired the ability to produce T lymphocyte reactions when incubated with reticulum cells in vitro. These results provide evidence about the role of the thymus reticulum in inducing immunocompetence of T lymphocytes.

A new approach for the direct enumeration of effector cells in populations containing cytotoxic T lymphocytes is described. Stable lymphocyte-target cell conjugates are formed during centrigugation at room temperature of mixtures containg cytotoxic lymphocytes and target tumor cells. Various types of lymphocyte-target cell conjugates can be formed, the composition of the conjugate being dependent upon the ratio of lymphocytes to target cells during the conjugation process. The number of conjugates formed is highest at equal lymphocyte and target cell concentrations. At least 35% of the BALB/c anti-EL4 peritoneal exudate lymphoid cells can conjugate to target cells and the majority of the conjugated lymphocytes are also killer cells. The variation in cytolytic activity of lymphoid cells with time following immunization and the differences in the lytic activity of immune lymphoid cells from various lymphoid organs are due to differences in the actual number of effector cells capable of conjugating with target cells.

The cell-mediated immune response of animals to a lethal syngeneic tumor was investigated by inoculating C57BL mice with Lewis lung carcinoma (3LL) cells T lymphocytes, obtained from the enlarged spleens of the tumor-bearing mice were found to be cytotoxic to 3LL target cells in vitro. However, we found that such spleen cells enhanced tumor growth in vivo when mice were injected with a mixture of spleen cells and tumor cells. Removal of T lymphocytes by treatment of the spleen cells with anti-Θ serum plus complement reduced the enhancement of tumor growth. Hence, the tumor enhancing cells, like the cytotoxic cells, appeared to be T lymphocytes. Removal of T lymphocytes from normal mice by adult thymectomy before tumor inoculation led to a reduction in the number of tumor metastases. Thus, enhancing T lymphocytes appear to exist in normal as well as in tumor-bearing mice. Investigation of this mechanism of tumor enhancement suggested that the enhancing T lymphocytes act as suppressor T cells inhibiting natural immune resistance to tumor growth.

The sequence of developmental processes of thymus-derived lymphocytes can be classified into two distinct stages. The first stage constitutes the appearance of lymphocytes in the thymus capable of recognizing antigenic signals. Such lymphocytes leave the thymus and colonize the spleen and lymph nodes. The second stage involves the response of thymocytes to antigenic signals. When a given cell-bound antigen triggers thymocytes capable of recognizing the antigen killer cells develop capable of lysing target cells possessing the same surface antigens that triggered their development. Studies indicated that both these stages are regulated by cell interactions. Results of the following studies are reported: in vitro induction of T-cell properties by thymus reticulum cells; interactions between thymocytes and the differentiation of effector cells; and control of the differentiation of effector lymphocytes by suppressor T-cells. (HLW)

The metastatic B16 mouse melanoma shows a low cell surface expression of H-2Kb and H-2Db class I antigens on cells of both the high-metastatic line B16-F10 and the low-metastatic line B16-F1. Similarly, newly generated clones of these lines, having different metastatic properties, all express low levels of major histocompatibility antigens. One of these clones, the high-metastatic F10.9, was transfected with H-2Kb genes to generate H-2Kb-expressing transfectants. The resulting clones showed reduced tumourigenicity and a low metastatic phenotype. Unlike the parental cells, H-2Kb-positive transfectants are potent inducers and sensitive targets of H-2Kb-restricted syngeneic cytotoxic T cells. Immunization of mice with H-2Kb-positive transfectants conferred protection against a subsequent challenge with Kb-positive transfectants but had only a small effect on growth and metastatic spread of parental cells.

To find out whether manipulation of H-2 expression on metastatic cells could alter their metastatic properties, we immunoselected in vivo H-2 antigen variants from a metastatic clone of the T10 sarcoma [originating in a (C57BL/6J X C3H.eB)FI mouse] and tested their metastatic capacity. The unselected metastatic cells (IE7) were previously found to express H-2Db and H-2Dk antigens, but they did not express the H-2K antigens of either parental haplotype. Transplantation of IE7 cells into C57BL/6J irradiated mice resulted in loss of H-2Dk expression and a reduction in H-2Db antigen density. Further transplantation of these cells into non-irradiated C57BL/6J mice led to a total loss of H-2 expression. The cells concomitantly lost their metastatic potency. Immunoselection of IE7 cells in C3H.eB irradiated and non-irradiated mice resulted in cells which were H-2Dk-positive but H-2Db-negative. Cells of these selected variants not only retained their metastatic potential, but in fact were far more metastatic than the unselected IE7 cells. Thus, changes in H-2 expression on tumor cells may alter their metastatic potential. In the case of T10 cells, H-2Dk expression seems to be directly involved in their metastatic capacity.

The control of the immunogenic antigen-presenting capacity of different subpopulations of thioglycollate-induced peritoneal macrophages had been investigated. The experiments revealed the existence of two major subpopulations of macrophages, only one of which was highly efficient in educating antigen-specific T cells. The other subpopulation, while highly phagocytic, was devoid of antigen-presenting capacity. Further analysis, using specific antisera directed at H-2I region gene products, revealed that the immunogenic antigen-presenting population expressed H-2I region-controlled membrane antigens. Searching for cellular elements which control the differentiation of this antigen-presenting macrophage subpopulation, it was found that its function was strictly controlled by T cells. T cell-deficient mice (nu/nu) failed to generate a functional antigen-presenting macrophage subpopulation. Transplantation of mature T lymphocytes to T cell-deprived mice restored the immunogenic function of their antigen-presenting macrophage subpopulation. Transplantation of mature T lymphocytes to T cell-deprived mice restored the immunogenic function of their antigen-presenting macrophages. The results obtained suggest the existence of heterogeneity of functions among macrophage subpopulations and add a new regulatory function for T cells.

When mouse spleen cells were stimulated by irradiated allogeneic cells in a mixed lymphocyte culture for 96 hours, allospecific suppressor cells were generated that could inhibit the replicative processes when transferred to another MLR. When the primed cells were purified on Ficoll and incubated alone for another 24 hours, they released into the medium a subcellular factor which could also suppress MLR allospecifically. Evidence is provided that the cells which generate this factor arise by cooperation of at least two types of T-lymphocytes, one of which is the progenitor of the suppressor cell and the other an essential accessory cell. Adherent cells were shown to be also essential for the priming of suppressor lymphocytes.

The immunogenic capacity of thioglycollate-induced peritoneal macrophages of adult splenectomized animals was compared to that of macrophages of sham-operated controls. Macrophages from splenectomized animals were found to be impaired in their function as antigen-presenting cells, both in the education of virgin initiator T lymphocytes and in the stimulation of antigen-specific T memory cells. Macrophages from splenectomized animals were also severely impaired in their phagocytic capacity, as assessed in an opsonin-dependent bacterial phagocytosis assay. However, they were not impaired in their ability to pinocytose soluble keyhole limpet hemocyanin. These results indicate that the spleen may play a decisive role in controlling the differentiation of peritoneal macrophages.

The effects of local tumor growth on the progression of lung metastasis of two mouse tumors of C57BL origin—the 3LL Lewis lung carcinoma and the B-16 melanoma—were studied. Excision of primary footpad grafts of 3LL or B-16 resulted in a significant increase in incidence and size of lung metastases. The incidence, but not the size, of lung metastases was found to be a function of size of the local tumor at time of excision. Reinoculation of tumor cells in the left footpad following excision of tumor from the right footpad suppressed the acceleration of growth of lung metastases and their increased incidence. Thus, the progression of the local tumor exerts an inhibitory effect on lung metastasis. The extent of inhibition of accelerated metastatic development by reinoculated tumor cells is a function of size of the cell inoculum or, in fact, of mass of the local tumor. The inhibitory effect of the local tumor on lung metastasis seems to be tumor-specific. 3LL metastases were not inhibited by local B-16 or EL4, neither was B-16 metastasis inhibited by 3LL. The specificity of interactions between the local tumor growth and its metastases may suggest the participation of immunological mechanisms in the control of lung metastasis. The involvement of the lymphoid system in the control of metastatic progression is also supported by the observation that total-body X-irradiation was associated with an increase in lung metastasis and that splenomegaly was observed in mice bearing the footpad tumor. Its excision resulted in the prevention of splenomegaly but reinoculation of the tumor, leading to metastatic suppression, was again associated with splenomegaly.

This study reports the production of clones of human killer T cells grown in the presence of TCGF4 following sensitization in MLC against (1) allogeneic cells, (2) autologous Bebv+ lymphocytes, or (3) autologous lymphoma cells. Sensitization against the tumor cells required the addition of macrophages. The expression of the cytotoxic activity of the cloned T lymphocytes required re-stimulation with the specific stimulator cells. The cytotoxic activity seemed to be MHC-restricted, since (1) cloned allosensitized CTL lysed their corresponding allogeneic targets, but did not lyse autologous Bebv+ cell or K562 cells; (2) cloned CTL sensitized against autologous Bebv+ cells lysed their autologous targets but not allogeneic Bebv+ targets or K562 cells; and (3) cloned CTL sensitized against autologous Burkitt lymphoma cells lysed their corresponding lymphoma targets or autologous Bebv+ targets but did not lyse allogeneic lymphoma cells or Bebv+ cells from the same allogeneic lymphoma cells or Bebv+ cells from the same allogeneic donors. The cloned CTL were homogeneous in expressing the OK T8 molecules and in being negative for T4, T10 or M1. At any given time, 25-45% of the cloned cells manifested lytic activity. The ultrastructural properties and cell surface OK T markers were different from those of cloned human NK cells. Emphasis is focused on the differences between the structural, functional and culture characteristics of CTL clones produced by direct isolation of MLC responder cells forming conjugates with specific target cells and those of clones from transformed T-cell lines or from T hybridomas.

SUMMARY Approximately 50% of fibrosarcomas induced with methylcholanthrene A were found to be defective in H-2 expression. In tumours which lack H-2K antigens, H-2 gene transfection was used to restore H-2K expression. The de novo expression of H-2K reduced tumorigenicity and abolished the formation of metastases in syngeneic mice. Expression of H-2K seems to render the tumour cells immunogenic and leads to effective recognition of the tumour cells by the host immune system.

Experiments are reported on immunotherapy of lethal lung metastasis by lymphocytes sensitized in culture against a syngenic tumor. We used the Lewis lung carcinoma (3LL) which had originated in a C57Bl mouse. C57Bl mice were footpad inoculated with tumor cells. Seven days liter, the tumor-bearing leg was amputated, and the animals were inoculated with syngenic lymphocytes which had been sensitized on monolayers of 3LL-tumor cells. The sensitized lymphocytes significantly reduced the development of lethal lung metastasis of the 3LL-tumor cells.

Isolation and characterization of individual functionally reactive cytotoxic T lymphocytes have been achieved. Peritoneal exudate cytotoxic lymphocytes were obtained from BALB/c mice injected with EL4 tumor cells. Lymphocyte tumor cell conjugation was promoted by centrifugation. Individual conjugates comprised of one lymphocyte bound to one tumor cell were isolated with a micropipette. The ultrastructure of isolated killer lymphocytes and the lysis of conjugated target cells were analyzed. The cytotoxic lymphocytes are small cells with an indented nucleus which is poor in peripheral chromatin and rich in rough nuclear sap. The cytoplasm contains one-membrane-bound lysosome-like granules and clusters of ribosomes, but no rough endoplasmatic reticulum. The Golgi apparatus is well developed. Direct evidence obtained at the single cell level shows that a single effector lymphocyte is required and sufficient for the destruction of a single target cell and that killer cells which have been responsible for the lysis of a given target cell can lyse a second and even a third time.

Two clones, E2-7.7 and E2-10.50, derived from two macrophage(MΨ)hybridomas, E2-7 and E2-10, have been studied. The first clone, E2-7.7, is Ia and Fc receptor (FcR) negative and manifests a strong antigen-presenting capacity. When we pulsed its cells in vitro with keyhole limpet hemocyanin (KLH) antigen and injected them into syngeneic animals, we found that as small a dose as 103 cells initiated an immune response in vivo. On the other hand, antigen-pulsed cells of the E2-10.50 clone, which are Ia- and FcR, were almost incapable of triggering immunity, even when injected at a dose of 105 cells. Thus, the two clones differ in their immunogenic capacity (both cellular and humoral immunity). In experiments aimed at testing the stimulation in vitro of primed lymph node (LN) cells by antigen-pulsed cells of these two hybridoma clones, we observed that E2-7.7 stimulated the unfractionated population of LN cells and the LN-derived population of T cells. The E2-10.50 cells stimulated only the unfractionated population of LN cells, but not the T cell population. Subsequent tests indicated that the E2-10.50 cells require an intermediate Ia accessory cell to present the antigen to the T lymphocytes. Analyzing the molecular structure of the MΨ hybridomas, we discovered that major histocompatibility complex (MHC) genes of the myeloma haplotype (H-2d), and of the splenic MΨ used for fusion (H-2k), which were not expressed in the parental myeloma or in the E2-10.50, were expressed in the E2-7.7. Thus, somatic cell fusion of MΨ resulted in the activation of suppressed genes of the myeloma partner. It appears that these antigens participate in controlling the immunogenic properties of the E2-7.7 clone. Testing the effects of interferons on the MΨ hybridomas, we observed that interferon-γ activated, at both the mRNA and the cell surface-antigen levels, the expression of H-2Dk, H-2Kd and H-2Dd in the E2-10.50 cells, but not in the E2-7.7. Consequently, interferon-γ augmented significantly antigen presentation by E2-10.50 but not by E2-7.7 cells. These two hybridoma clones might represent two distinct subsets of normal MΨ, manifesting two different sets of functional properties. Having described earlier the E2-10.20 clone that takes up, but does not present antigen, we may in fact be facing three types of MΨ hybridomas, representing three functional subpopulations of MΨ.

Intravenous inoculation of the AKR mouse-strain-derived BW lymphoma into CBA recipients resulted in a case of liver metastasis; cells derived from this metastatic nodule were termed BW-Li cells. BW-Li cells, upon reinoculation, generated metastases in the spleen, liver, kidney and ovaries in 100% of CBA recipients. Furthermore, BW-Li cells, in contrast to BW cells, were found to infiltrate in vitro monolayers of hepatocytes, thus confirming their inherent invasive potential. Analysis of the alloantigenic phenotype of BW-Li cells revealed that such cells were Thy 1.1+, Thy 1.2+, Lyt 1.2+, Lyt 1.1-, Lyt 2- and H-2Dk+, as compared to BW cells which exhibited the membrane phenotype Thy 1.1+, Thy 1.2-, Lyt 1.2-, Lyt 1.1-, Lyt 2-, H-2Dk-. BW-Li cells also differed functionally from BW cells since these cells secreted IL-2 upon stimulation with Concanavalin A. BW tumor transplantation experiments were repeated in a semi-allogeneic F1 strain combination, i.e. (AKR X CBA)F1, and again a case of massive liver metastasis was observed. Cells derived from these liver metastases (termed BW-O-Li) manifested an invasive and metastatic potential similar to that of BW-Li cells. Furthermore, BW-O-Li cells secreted IL-2 upon stimulation with Con A and manifested the following alloantigenic phenotype: Thy 1.1+, Thy 1.2+, Lyt 1.2+, Lyt 1.1-, Lyt 2-, H-2Dk+ and H-2Kk+. These results indicate that BW-Li and BW-O-Li cells are functional T-cell hybrids which express T-cell markers derived from BW cells and Thy 1.2+ CBA host cells. The acquisition of host-derived T-cell properties may have led to the expression of metastatic and invasive capabilities. From these results we conclude that the acquisition of metastatic properties following somatic cell fusion with normal lymphoreticular cells may represent a mechanism for tumor progression in vivo.