Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities.

Elevated levels of p130(Cas) (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130(Cas) protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130(Cas)-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130(Cas) exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130(Cas) on cell biology and therefore will be the target of future studies.

Towards neuroimmunotherapy for cancer: the neurotransmitters glutamate, dopamine and GnRH-II augment substantially the ability of T cells of few head and neck cancer patients to perform spontaneous migration, chemotactic migration and migration towards the autologous tumor, and also elevate markedly the expression of CD3zeta and CD3epsilon TCR-associated chains.

In previous studies we found that several Neurotransmitters and Neuropeptides among them: Glutamate, Dopamine, Gonadotropin-releasing-hormone (GnRH) I and II, Somatostatin, CGRP and Neuropeptide Y, can each by itself, at low physiological concentration (~10 nM) bind its receptors in human T cells and trigger several key T cell functions. These findings showed that the nervous system, via Neurotransmitters and Neuropeptides, can 'talk' directly to the immune system, and stimulate what we coined 'Nerve-Driven Immunity': immune responses dictated by the nervous system. In various human cancers, the immune system of the patients, and their T cells in particular, are not functioning well enough against the cancer due to several reasons, among them the suppressive effects on the immune system induced by: (1) the cancer itself, (2) the chemotherapy and radiotherapy, (3) the ongoing/chronic stress, anxiety, depression and pain felt by the cancer patients. In Head and Neck Cancer (HNC), 5-year survival rate remains below 50%, primarily because of local recurrences or second primary tumors. Two-thirds of HNC patients are diagnosed at advanced clinical stage and have significantly poorer prognosis. Most HNC patients have multiple severe immunological defects especially in their T cells. A major defect in T cells of patients with HNC or other types of cancer is low CD3zeta expression that correlates with poor prognosis, decreased proliferation, apoptotic profile, abnormal cytokine secretion and poor abilities of destructing cancer cells. T cells of cancer patients are often also unable to migrate properly towards the tumor. In this study we asked if Glutamate, Dopamine or GnRH-II can augment the spontaneous migration, chemotactic migration and towards autologous HNC migration, and also increase CD3zeta and CD3epsilon expression, of peripheral T cells purified from the blood of five HNC patients. These HNC patients had either primary tumor or recurrence, and have been already treated by surgery and/or radiotherapy and/or chemotherapy without satisfactory outcomes. We found that Glutamate, Dopamine and GnRH-II, each by itself, at 10 nM, and during 30 min incubation only with the peripheral T cells of the HNC patients increased substantially their: (1) spontaneous migration (up to 4.4 fold increase), (2) chemotactic migration towards the key chemokine SDF-1 (up to 2.3 fold increase), (3) migration towards the autologous HNC tumor removed surgically ~48 h earlier in a pre-planned operation (up to 3.5 fold increase). Each of the Neurotransmitters even 'allowed' the T cells of one HNC patient to overcome completely the suppressive anti-migration effect of his autologous tumor, (4) cell surface CD3zeta expression (up to 4.3 fold increase), (5) cell surface CD3epsilon expression (up to 1.9 fold increase). If the absolutely essential larger scale subsequent studies would validate our present findings, Glutamate, Dopamine and GnRH-II could be used for a completely novel indication: adoptive T cell immunotherapy for some patients with HNC and maybe also other types of cancer. We coin here a novel term-'Neuroimmunotherapy' for this new form of T cell immunotherapy, based on the direct activation of the patient's own T cells by Neurotransmitters. Such 'Neuroimmunotherapy' could be reduced to practice by rather simple, painless and repeated/periodical removal of peripheral T cells from the cancer patients, activating them ex vivo for 30 min by either Glutamate, Dopamine or GnRH-II, and infusing them back to the patients by intravenous and/or intratumoral injection. The 'rejuvenated' Neurotransmitter-treated T cells are expected to have significantly improved abilities to reach and eradicate the cancer, and also combat infectious organisms that cancer patients often suffer from. Since the T cells are autologous, since the Neurotransmitters are physiological molecules, and since the ex vivo 'parking period' is very short, such Neuroimmunotherapy is expected to be very safe.

Benefits of a continuous therapy for cancer patients with a novel adoptive cell therapy by cascade priming (CAPRI).

A growing body of evidence shows that immune cells are pivotal in the fight against cancer. First, association studies have identified immune-protective immune response genes against cancer. Second, the presence of immune cells in the respective malignant tumor correlated with a better prognosis for the patients. Third, adoptive cell therapy (ACT) showed, in recent reports, an efficient reduction or even cure for malignant tumors. The focus of this review is a novel in vitro ACT technique, using the patient's cascade-primed immune cells. The cascade-priming procedure stimulates APCs from the peripheral blood. Stimulated APCs digest and present tumor material better and differentiate naive cytotoxic T-lymphocyte effector cells against the patient's cancer. The principle and the impressive results of the cascade-primed immune cell treatment in patient case series is compared with the ACT concepts of lymphokine-activated killer, macrophage-activated killer, macrophage-activated killer-dendritic cell, cytokine-induced killer and tumor-infiltrating lymphocyte methods.

Prolongation of life by adoptive cell therapy with cascade primed immune cells in four patients with non-small cell lung cancer stages IIIB and IV and a pancoast tumor: a case series.

INTRODUCTION: Despite newer treatment modalities, few patients with non-small cell lung cancer in stages IIIB and IV survive the median of one year. We present four patients with non-small cell lung cancer treated with an adjuvant therapy with cascade primed immune cells. The in vitro stimulated expression of cancer information on the patients' monocytes matures and activates T lymphocytes to destroy cancer cells. The cascade primed immune cell therapy significantly improved the quality of life and the lifespan of all four patients; thus far, three patients survived 40, 55 and 120 months, respectively; and one patient died 39 months after diagnosis. CASE PRESENTATION: Patient 1, stage IV (T4N2M1): The adenocarcinoma of the 67-year-old German Caucasian man infiltrated into the mediastinal lymph nodes and iliosacral bones. Chemotherapy modalities were started immediately after diagnosis of cancer, and cascade primed immune cell therapy one year later. The patient survived 39 months.Patient 2, stage IV (T3N3M1a): The 62-year-old German Caucasian woman presented with adenocarcinoma of the lower lobe with infiltrated lymph nodes of the mediastinum and malignant pleural effusion. Chemotherapy, radiation and the cascade primed immune cell therapy were administered together. The patient is still alive after 40 months.Patient 3, stage IIIB (T4N1-2M0): The 75-year-old German Caucasian woman presented with an undifferentiated tumor and a separate tumor nodule in the ipsilateral lobe. The patient received only cascade primed immune cell therapy after tumor resection and has survived for the last 55 months.Patient 4, pancoast tumor (IIIB, T3N3M0): The 77-year-old German Caucasian man presented with an undifferentiated tumor that infiltrated the lymph nodes, the clavicle, one rib and the plexus brachialis. In addition to chemotherapy and radiation, cascade primed immune cells were administered every weekday for one year. After four months, no living tumor cell was detected in the resected lung, the lymph nodes or the bone material. The patient is still alive after 120 months. CONCLUSIONS: The novel adoptive cell therapy with cascade primed immune cells significantly increased the survival rate and maintained the quality of life for four patients with non-small cell lung cancer in stages IIIB and IV. Our findings indicate that tumor resection, chemotherapy and radiation appear to support the cascade primed immune cell therapy.

Paradoxical downregulation of HLA-A expression by IFNγ associated with schizophrenia and noncoding genes.

Neuronal MHC/HLA regulates the synapses of the central nervous system (CNS). The expression of MHC/HLA is, in turn, regulated by immune cytokines. We were therefore interested in the regulation of schizophrenia-associated HLA antigens, specifically their regulation of expression by interferons. We had previously observed a moderately increased frequency of HLA-A10 expression in schizophrenic patients. While searching for the "true" disease gene near the HLA-A gene, we discovered that homozygosity of the HLA-J M80469 pseudogene allele, in combination with HLA-A10 or HLA-A9, was associated with a high risk of schizophrenia (HLA-A10 relative risk = 29.33, p = 0.00019, patients N = 77, controls N = 214). The allele HLA-J M80468, which codes for interferon-inducible mRNA, conferred protection on carriers of HLA-A9 and HLA-A10 (HLA-A10 relative risk = 0.022, p = 0.00017). Functional analysis revealed that interferon γ (IFNγ) downregulated the expression of HLA-A9 and HLA-A10 in monocytes from HLA-J M80469 homozygous patients but not from carriers of the HLA-J M80468 allele. This is the first demonstration of an inverse effect of IFNγ on HLA expression that is associated with non-coding gene variants and schizophrenia. Our findings suggest that the interferons secreted during acute and chronic infections may interfere in synaptic regulation via neuronal HLA and that this disturbance in synaptic regulation may induce the symptoms of mental illness.

Responsiveness of a patient in a persistent vegetative state after a coma to weekly injections of autologous activated immune cells: a case report.

INTRODUCTION: An 82-year-old Caucasian woman had remained in a persistent vegetative state after a coma of seven months duration, which occurred after a stroke with hemiplegia, nine years previously. The persistent vegetative state could be reversed in part by weekly injections with activated immune cells. After therapy, our patient responded to commands in addition to regaining spontaneous movements of both arms and the ability to swallow. This is the first report on the treatment with activated immune cells of a patient in a persistent vegetative state after a coma. CASE PRESENTATION: An 82-year-old Caucasian woman presented with a persistent vegetative state subsequent to a coma. She retained respiratory and autonomic functions. As contact was not possible, physiotherapy was passive. Her skin was yellowish, and our patient did not move by herself. Vomiting repeatedly resulted from tube feeding. After a once-weekly treatment with activated immune cells sampled from our patient's blood and activated in vitro, several of her functions gradually returned. Our patient opened her eyes in the requested direction and turned her head toward people entering the room. She 'supported' nursing efforts, as the nurse noted a loss of spastic motions. The strength in both her arms returned, and she spontaneously moved her arm on the side experiencing hemiplegia. After three months, our patient could stick out her tongue upon demand. Finally, the swallow reflexes of our patient started to return. However, tube feeding was continued, and our patient died after aspiration of vomit following a feeding. CONCLUSION: The success of treatment with autologous activated immune cells in this patient may have resulted from the production of neuroactive substances, such as neurotrophin-3 and brain-derived neurotrophic factor, by activated immune cells. The deterioration of our patient could be reversed, as demonstrated by the restoration of motor strength in her hemiplegic side. In addition, our patient was able to induce motor responses upon request. It seems reasonable to conclude that activated immune cells may improve the chronic vegetative state in some patients.

The LMP7-K allele of the immunoproteasome exhibits reduced transcript stability and predicts high risk of colon cancer.

Destruction of cancer cells by cytotoxic T lymphocytes depends on immunogenic tumor peptides generated by proteasomes and presented by human leukocyte antigen (HLA) molecules. Functional differences arising from alleles of immunoproteasome subunits have not been recognized so far. We analyzed the genetic polymorphism of the immunoproteasome subunits LMP2 and LMP7 and of the transporters associated with antigen processing (TAP1 and TAP2) in two independently collected panels of colorectal carcinoma patients (N(1) = 112, N(2) = 62; controls, N = 165). High risk of colon cancer was associated with the LMP7-K/Q genotype (OR = 8.10, P = 1.10 × 10(-11)) and low risk with the LMP7-Q/Q genotype (OR = 0.10, P = 5.97 × 10(-13)). The basis for these distinct associations of LMP7 genotypes was functionally assessed by IFN-γ stimulation of colon carcinoma cell lines (N = 10), followed by analyses of mRNA expression of HLA class I, TAP1, TAP2, and LMP7, with real-time PCR. Whereas induction of HLA-B, TAP1, and TAP2 was comparable in all cell lines, transcript amounts of LMP7-Q increased 10-fold, but of LMP7-K only 3.8-fold. This correlated with a reduced transcript stability of LMP7-K (t(1/2) ≈ 7 minutes) compared with LMP7-Q (t(1/2) ≈ 33 minutes). In addition, LMP7-Q/Q colon carcinoma cells increased (the peptide based) HLA class I surface expression significantly after IFN-γ stimulation, whereas LMP7-Q/K and LMP7-K/K carcinoma cells showed minimal (<20%) changes. These results suggest that the presence of LMP7-K can reduce the formation of immunoproteasomes and thus peptide processing, followed by reduced peptide-HLA presentation, a crucial factor in the immune response against cancer.

Inhibition of pleural metastasis of collecting duct carcinoma of the kidney by modified cytokine-induced killer cells: A case report and review of the literature.

Collecting duct carcinoma (CDC) or Bellini duct carcinoma of the kidney is a rare, but highly aggressive renal epithelial malignancy, with an extremely poor prognosis. Modified cytokine-induced killer (mCIK) cells were injected into the pleural cavity to treat pleural metastasis of CDC. The patient, a 33-year-old male, was admitted to hospital for further treatment for severe pleural metastasis of CDC. We cured the pleural metastasis through intrapleural infusion with mCIK cells. After receiving this innovative treatment, the patient exhibited a positive response: the cough, dyspnea, chest distress and thoracalgia were evidently relieved, while the pleural fluid became clear after exhibiting haematodes and its level decreased significantly. The patient achieved partial success. This novel immunotherapy method is a promising treatment for patients with refractory pleural metastasis.

High risk of schizophrenia and other mental disorders associated with chlamydial infections: hypothesis to combine drug treatment and adoptive immunotherapy.

Many microbial factors have been implicated as pathogenic factors in mental disorders. Occurrence of such microbial factors also in the mentally unaffected population raised skepticism against such findings, although each microbial factor may cause mental problems only in some individuals, depending on the individual's immunogenetic disposition. Skepticism against the role of infection in schizophrenia was also fostered by the low impact of antiinfections treatment on the course of disease progression in schizophrenia. We discovered previously that neurotrophins like neurotrophin3 (NT-3) and brain-derived neurotrophic factor (BDNF), involved in processes of neuroplasticity, are also secreted by immune cells, but only by subpopulations of immune cells. Therefore, infection of the immune cell subpopulation, specialized in secreting BDNF, or of another subpopulation, specialized in secreting NT-3, could distort communication of immune cells with the central nervous system (CNS). Chlamydiaceae could cause disbalancement of immune cell sub-populations and, in some individuals with a vulnerable disposition, symptoms of mental illness. Based on previous observations of persisting IgA titers in some patients with mental illness we hypothesize that the intracellular parasites Chlamydiaceae are main pathogenic factors in schizophrenia. We hypothesize furthermore that antiinfectious treatment has to be accompanied by adoptive immunotherapy because antibiotics alone will not restore the balance of immune subpopulations. Our hypothesis is supported by examination of patients with schizophrenia and other mental disorders. Using nested PCR we found a significant prevalence of the intracellular parasites Chlamydophila psittaci, C. pneumoniae and Chlamydia trachomatis (9/18, 50%), as compared to controls (8/115, 6.97%) (chi(2)=25.86, Fisher's exact p two-tailed=5x10(-5)). Treatment with in vitro-activated immune cells together with antibiotic modalities showed sustained mental improvements in patients that did not depend on treatment with antipsychotic drugs. Future controlled studies including sham treatment of patients have to be carried out to prove our hypotheses.