Therapeutic T cells induce tumor-directed chemotaxis of innate immune cells through tumor-specific secretion of chemokines and stimulation of B16BL6 melanoma to secrete chemokines

Background

Given the considerable toxicity and modest benefit of adjuvant chemotherapy for non-small cell lung cancer (NSCLC), there is clearly a need for new treatment modalities in the adjuvant setting. Active specific immunotherapy may represent such an option. However, clinical responses have been rare so far. Manipulating the host by inducing lymphopenia before vaccination resulted in a magnification of the immune response in the preclinical setting. To evaluate feasibility and safety of an irradiated, autologous tumor cell vaccine given following induction of lymphopenia by chemotherapy and reinfusion of autologous peripheral blood mononuclear cells (PBMC), we are currently conducting a pilot-phase I clinical trial in patients with NSCLC following surgical resection. This paper reports on the first clinical experience and evidence of an immune response in patients suffering from NSCLC.

Methods

NSCLC patients stages I-IIIA are recruited. Vaccines are generated from their resected lung specimens. Patients undergo leukapheresis to harvest their PBMC prior to or following the surgical procedure. Furthermore, patients receive preparative chemotherapy (cyclophosphamide 350 mg/m² and fludarabine 20 mg/m² on 3 consecutive days) for induction of lymphopenia followed by reconstitution with their autologous PBMC. Vaccines are administered intradermally on day 1 following reconstitution and every two weeks for a total of up to five vaccinations. Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is given continuously (at a rate of 50 μg/24 h) at the site of vaccination via minipump for six consecutive days after each vaccination.

Results

To date, vaccines were successfully manufactured for 4 of 4 patients. The most common toxicities were local injection-site reactions and mild constitutional symptoms. Immune responses to chemotherapy, reconstitution and vaccination are measured by vaccine site and delayed type hypersensitivity (DTH) skin reactions. One patient developed positive DTH skin tests so far. Immunohistochemical assessment of punch biopsies taken at the local vaccine site reaction revealed a dense lymphocyte infiltrate. Further immunohistochemical differentiation showed that CD1a+ cells had been attracted to the vaccine site as well as predominantly CD4+ lymphocytes. The 3-day combination chemotherapy consisting of cyclophosphamide and fludarabine induced a profound lymphopenia in all patients. Sequential FACS analysis revealed that different T cell subsets (CD4, CD8, CD4CD25) as well as granulocytes, B cells and NK cells were significantly reduced. Here, we report on clinical safety and feasibility of this vaccination approach during lymphoid recovery and demonstrate a patient example.

Conclusion

Thus far, all vaccines were well tolerated. The overall trial design seems safe and feasible. Vaccine site reactions associated with infusion of GM-CSF via mini-pump are consistent with the postulated mechanism of action. More detailed immune-monitoring is required to evaluate a potential systemic immune response. Further studies to exploit homeostasis-driven T cell proliferation for the induction of a specific anti-tumor immune response in this clinical setting are warranted.     

Maximizing phenotype constraint and extracellular matrix production in primary human chondrocytes using arginine–glycine–aspartate concentration gradient hydrogels

New systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using the limited cell numbers obtained from primary human sources. Peptide functionalized hydrogels possessing continuous variations in physico-chemical properties are an efficient three-dimensional platform for studying several properties simultaneously. Herein, we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system possessing a gradient of arginine–glycine–aspartic acid peptide (RGD) concentrations from 0 mM to 10 mM. The system is used to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance and extracellular matrix (ECM) production to the gradient hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with higher RGD concentrations, while regions with lower RGD concentrations maintained cell number and phenotype. Over three weeks of culture, hydrogel regions containing lower RGD concentrations experience an increase in ECM content compared to regions with higher RGD concentrations. Variations in actin amounts and vinculin organization were observed within the RGD concentration gradients that contribute to the differences in chondrogenic phenotype maintenance and ECM expression.     

Effects of Echinostoma caproni miracidia dose on the neutral and polar lipids of Biomphalaria glabrata as determined by high-performance thin-layer chromatography

The effects of a 5 versus 25 miracidia exposure of Echinostoma caproni on the lipid composition of Biomphalaria glabrata was studied using high performance thin layer chromatography (HPTLC)-densitometry. A 50 miracidia dose was not used because such a high level of exposure caused severe snail mortality by 3 weeks post-exposure (PE). Lipids were determined in the digestive-gland gonad complex (DGG) of the exposed snails and in the uninfected matched controls at 2 and 4 weeks PE. Extraction of lipids from DGGs was carried out by the Folch method with chloroform-methanol (2:1), and extracts were analyzed on Analtech HPTLC-HLF pre-adsorbent silica gel plates with measurement of separated bands using a CAMAG Scanner 3. For neutral lipids the mobile phase was petroleum ether-diethyl ether-glacial acetic acid (80:20:1) and the detection reagent was 5% ethanolic phosphoric acid, and for polar lipids chloroform-methanol-deionized water (65:25:4) mobile phase and 10% cupric sulfate in 8% phosphoric acid detection reagent were used. No significant differences in the concentrations of free sterols, free fatty acids, triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine were seen at 2 weeks PE in any of the groups. At 4 weeks PE, the free fatty acid concentration increased significantly in the snails exposed to 25 miracidia compared to that of the 5 miracidia/snail group or the controls. Elevation of the free fatty acid fraction in the high dose snail group suggested that some changes occurred in the lipid metabolism of the snails in that group as a function of miracidia dose.     

Identification of rare copy number variants in high burden schizophrenia families

Over the last years, genome‐wide studies consistently showed an increased burden of rare copy number variants (CNVs) in schizophrenia patients, supporting the “common disease, rare variant” hypothesis in at least a subset of patients. We hypothesize that in families with a high burden of disease, and thus probably a high genetic load influencing disease susceptibility, rare CNVs might be involved in the etiology of schizophrenia. We performed a genome‐wide CNV analysis in the index patients of eight families with multiple schizophrenia affected members, and consecutively performed a detailed family analysis for the most relevant CNVs. One index patient showed a DRD5 containing duplication. A second index patient presented with an NRXN1 containing deletion and two adjacent duplications containing MYT1L and SNTG2. Detailed analysis in the subsequent families showed segregation of the identified CNVs. With this study we show the importance of screening high burden families for rare CNVs, which will not only broaden our knowledge concerning the molecular genetic mechanisms involved in schizophrenia but also allow the use of the obtained genetic data to provide better clinical care to these families in general and to non‐symptomatic causal CNV carriers in particular. © 2013 Wiley Periodicals, Inc.     

Morphologic features of endometriosis in various types of cytologic specimens

Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. This study evaluates the cytomorphologic features of endometriosis in various cytologic specimen types [fine‐needle aspiration (FNA), effusion cytology (EF), touch imprint (ToP), and cervical smear (PAP)], and assesses the key elements helpful in recognizing this lesion. A total of 18 cases (8 FNA, 4 EF, 5 ToP, and 1 PAP) of cytologically diagnosed and histologically/clinically confirmed endometriosis diagnosed between 1988 and 2006 comprises the material for this study. The morphologic features evaluated of the three components included: cellularity, presence of sheets of glandular cells, three‐dimensional (3D) glandular clusters, tubular structures, single cells, syncytial groups of stromal cells, stromal cells entrapped within basement membrane (BM)‐like material, cytologic atypia, presence of mitotic figures, and hemosiderin‐laden histiocytes. Endometrial glands, stroma, and hemosiderin‐laden histiocytes were all identified in 14/18 (77.8%) cases. FNA specimens were more cellular than that of both EF and ToP specimens. Tubular structures, 3D glandular clusters, stromal cells entrapped in BM and syncytial stromal groups were more common in FNAs, and ToPs compared with the EFs. The ratio of the endometrial glandular and stromal cells was similar in all specimen types. Atypia and mitotic figures were rarely encountered. Diagnosis of endometriosis could be made independently on either smears/ThinPrep^? slides or on cell blocks in all cases where these preparations were available. On follow up, none of the patients developed malignancy. Endometriosis can be reliably and safely diagnosed in various cytologic materials. Cytologic atypia is uncommon. Components of endometriosis could show minor morphologic alterations in different specimen types. Diagn. Cytopathol. 2013;41:936–942. © 2013 Wiley Periodicals, Inc.     

Neonatal Liver Disease Associated with Placental Transfer of Anti-mitochondrial Antibodies

Background: Anti-mitochondrial antibody is the diagnostic hallmark of primary biliary cirrhosis. Its role in the aetiology of primary biliary cirrhosis is controversial. Methods: Two cases of neonatal hepatitis seropositive for anti-mitochondrial antibody are described. Anti-mitochondrial antibody Ig isotype and epitopic specificity were investigated by immunofluorescence and enzyme immunoassays. Results: In both infants anti-mitochondrial antibody was of the G class, mainly G1 and G3 subclasses, and reacted with two synthetic peptides reproducing major M2 epitopic regions: inner lipoyl domain pyruvate dehydrogenase complex (PDC)-E2_162-176 and PDC-E3 binding protein (PDC-E3BP)_86-100. One infant also reacted with outer lipoyl domain PDC-E2_35-49, and 2-oxoglutarate dehydrogenase complex (OGDC)-E2_99-113. An identical pattern of reactivity was present in their mothers, indicating the maternal origin of the antibodies. Anti-mitochondrial antibody disappeared in the infants with the disappearance of the liver pathology. Conclusions: The simultaneous disappearance of hepatitis and anti-mitochondrial antibody in the infants suggests a possible causal link between the two.     

Topography and ultrastructure of the tegument of Deropristis inflata Molin, 1859 (Digenea: Deropristidae), a parasite of the European eel Anguilla anguilla (Osteichthyes: Anguillidae)

The tegumental ultrastructure of the intestine fluke Deropristis inflata was studied using scanning and transmission electron microscopy. The surface of the tegument was covered by transverse cytoplasmic ridges from which protrude numerous thorn-like spines showing crenelated tips on the posterior part. Spines were arranged in staggered rows. Cobblestone-like units of the tegument were observed on a semicircle-shaped formation over the oral sucker. A tegumental excrescence was observed in the dorsal anterior side of the fluke. Ultrastructural study revealed that the tegument of D. inflata had a typical syncytial organization with a distal cytoplasm lying over a basal matrix and cytons. Cytoplasmic bridges allowed transit of secretory vesicles and granules packed in gland cells. Two types of sensory structures were examined. Type 1 sensory receptor was a button-like uniciliated papilla mounted on a folded tegumental base and surrounded by cytoplasmic ridges. This receptor consisted of a nerve bulb and a cilium that extended from a centriole. Type 2 sensory receptor was a smooth bulb-like non-ciliated papilla. It was only recovered on the ventral sucker. This receptor consisted of a nerve bulb enclosing an ovoid electron-dense structure. For both receptors, the nerve bulbs contained numerous mitochondria, nerve fibers, and electron-lucent material. Particular distributions of the sensory receptors were observed with a concentration on the anterior third of the body around the oral and ventral suckers. Diagrams were made to help in understanding the nature of these structures.     

Analysis of Human Bone Sialoprotein in Normal and Pathological Tissues using a Monoclonal Antibody (BSP 1.2 mab)

Bone sialoprotein (BSP), a phosphorylated and sulphated glycoprotein that is expressed by mineralized connective tissues is also produced in tumors that metastasize to bone. To facilitate studies of BSP expression in normal and pathological human tissues a monoclonal antibody (BSP 1.2 mab) was raised against human bone BSP. BSP 1.2 mab was shown by ELISA assays to recognize the epitope “DEYSY” (amino acids 279–283) that is conserved in mammalian BSP sequences. However, whereas the antibody recognized recombinant BSPs expressed in bacteria, it did not recognize native forms of rat or pig BSP in which the first tyrosine of the DEYSY peptide sequence appears to be modified. Immunostaining of embryonic human tibiae and calvariae with BSP 1.2 mab showed strong reaction in osteoblasts and osteocytes with relatively weak staining of the bone matrix, suggesting that the BSP 1.2 mab epitope is partially masked in the bone matrix. BSP 1.2 mab also stained osteosarcoma cells and normal trophoblastic cells in the placenta in areas of microcrystalline deposits. Cancer cells in primary breast tumors, lymph nodes, and secondary bone metastases from individual patients were stained strongly by BSP 1.2 mab. Although BSP 1.2 mab also stained breast cancer carcinoma cell lines and SaOS2 osteosarcoma cells, biosynthesis of radiolabelled BSP could not be demonstrated in breast cancer cells. Notably, the staining of BSP in the breast cancer cells was diffuse contrasting the punctate staining, typical of secreted proteins, in SaOS2 cells. These studies, therefore, have identified a unique epitope in human BSP recognized by a monoclonal antibody, BSP 1.2 mab, which can be used for the unequivocal identification of BSP in normal and pathological human tissues.     

A continuous decline in menarcheal age in Denmark

We report a renewed decline in mean menarcheal age in a large Danish sample after a period with a halt in the trend towards earlier age at menarche in many North European countries. In our study based on retrospective data from six different samples constituting 42784 women, we find a continuously declining mean menarcheal age in Denmark among women born in the years 1964-1973. In a sample of textile workers born in the years 1939-1968 (n = 12605) we find a 1 year higher mean menarcheal age. This indicates that menarcheal age is still delayed in certain groups in Denmark. This leaves the possibility that the menarcheal age could fall even further in the future.