Downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial DNA as a potential regulator.

Antibacterial peptides are active defense components of innate immunity. Several studies confirm their importance at epithelial surfaces as immediate barrier effectors in preventing infection. Here we report that early in Shigella spp. infections, expression of the antibacterial peptides LL-37 and human beta-defensin-1 is reduced or turned off. The downregulation is detected in biopsies from patients with bacillary dysenteries and in Shigella- infected cell cultures of epithelial and monocyte origin. This downregulation of immediate defense effectors might promote bacterial adherence and invasion into host epithelium and could be an important virulence parameter. Analyses of bacterial molecules causing the downregulation indicate Shigella plasmid DNA as one mediator.

Evaluation of ex vivo expanded human NK cells on antileukemia activity in SCID-beige mice.

The possibility of using natural killer (NK) cells in treatment of human hematological malignancies has increased in recent years. One factor contributing to this is the introduction of new methods for ex vivo generation of enriched populations of clinical grade NK cells. The objective of the present study was to evaluate the safety and efficacy of human ex vivo expanded clinical grade NK cells against K562 leukemia cells in severe combined immunodeficiency disease (SCID)-beige mice. Irradiated SCID-beige mice were injected intravenously (i.v.) with K562 leukemia cells. Following leukemia cell injection, mice were injected with ex vivo expanded human NK cells. NK cells were followed in vivo and mice monitored for survival from leukemia. Administration of these ex vivo expanded clinical grade NK cells was safe and prevented leukemia development. In conclusion, these results imply possibilities for the use of this NK cell preparation in treatment trials of human hematological malignancies and possibly other forms of cancer.

Gap junctions promote the bystander effect of herpes simplex virus thymidine kinase in vivo.

Transfer of the herpes simplex thymidine kinase gene (HSVtk) into tumor cells followed by the administration of ganciclovir (GCV) provides a potential strategy for the treatment of some malignancies. During GCV treatment, not only the cells that express the HSVtk gene are killed but also frequently neighboring tumor cells that are not genetically altered. This has been called the "bystander effect." Although the mechanism of the bystander effect in vivo remains elusive, our results suggest that gap junction formation between neighboring cells is an important contributing factor. The C6 rat glioma cell line, which exhibits a low level of intercellular communication by gap junctions and connexin43 (Cx43)-transfected clones of this cell line forming gap junctions from a moderate level (Cx43-12 and Cx43-14) to a high level (Cx43-13), were transduced with HSVtk. Transduced and nontransduced cells were mixed in various concentrations and then cultured in vitro or injected s.c. into C.B-17/SCID-beige mice followed by i.p. injections of GCV. Cx43-transfected clones showed a significant increase of the bystander effect compared with the less coupled C6 parental cell line. In 11 of 12 mice injected with cells of Cx43-transfected clones, no tumors were seen at the inoculation site when a mixture of 50% HSVtk-negative and HSVtk-positive cells was used. Moreover, in mice injected with cells of clone Cx43-13, which exhibits the highest intercellular communication, tumors were frequently undetectable at the inoculation site when using mixtures of 75% HSVtk-negative and 25% HSVtk-positive cells, and even mixtures containing 5% HSVtk-positive cells of Cx43-transfected clones showed tumor size reduction. All animals in control groups (n = 26) developed large tumors at every injection site. These results demonstrate that gap junctions are an important component in mediating the bystander effect in vivo.

Clonability and tumorigenicity of human epithelial cells expressing the EBV encoded membrane protein LMP1.

Two isolates of the EBV-LMP1 gene were compared for their ability to induce phenotypic changes in a non-tumorigenic human keratinocyte line, Rhek-1, immortalized with an adenovirus 12-SV40 hybrid virus. One isolate, designated B-LMP1, was derived from B95-8, a B-cell line of marmoset origin, that carries a viral strain from a mononucleosis patient. The other, designated C-LMP1, originated from a nude mouse passaged Chinese NPC tumor, CAO. Both types of transfectants were less serum dependent than the non-transfected and the vector-transfected controls. The ability to grow on low serum increased with increasing LMP1 expression. All transfectants were more highly clonable than the non-transfected or vector-transfected controls. Clonability in soft agarose increased with increasing LMP1 expression. Nine of 24 C-LMP1 transfectants produced tumors in SCID mice. Seven of them grew invasively into the surrounding tissue. Only one of 12 B-LMP1 transfected Rhek-1 clones was tumorigenic. It did not grow invasively. All tumorigenic transfectants expressed LMP1 at high or moderate levels. All tumors were found to express LMP1. Transfectants with low LMP1 expression did not produce tumors. The untransfected Rhek-1 cells and six vector control clones failed to produce tumors.

Clonal immunoglobulin gene rearrangements in primary mediastinal clear cell lymphomas.

Cells from two patients with primary mediastinal tumors of clear cell type were characterized by immunological and molecular biological techniques. In both cases a B cell immunophenotype was suggested by the positive staining for the B1 (CD20), B4 (CD19), Leu 14 (CD22), as well as staining with other B monoclonal antibodies by immunohistochemistry. However, no definitive evidence for the expression of Ig light or heavy chains at the protein level was found. Southern blot analysis of Ig heavy and light chain gene rearrangements revealed clonal B cell populations in both cases. There was no indication of a somatic joining of T cell receptor (TCR) genes using probes for TCR beta and or TCR gamma genes. Thus, our results suggest a clonal B cell origin of clear cell lymphomas in the mediastinum.

Expansion of natural killer (NK) and natural killer-like T (NKT)-cell populations derived from patients with B-chronic lymphocytic leukemia (B-CLL): a potential source for cellular immunotherapy.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world. It is currently an incurable disease, making new treatment options such as immunotherapy desirable. Monoclonal antibodies (Mabs) to surface antigens of the tumor cell is one option. Administration of cytotoxic cells such as natural killer (NK) and natural killer-like T (NKT) cells expanded in vitro might be a useful treatment modality alone or in combination with MAbs. A limiting step in the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Here, we report the feasibility of expanding populations of the human killer cells, CD3-CD56+ NK and CD3+CD56+ NKT cells, from peripheral blood mononuclear cells (PBMCs) of B-CLL patients. The influence of tumor B cells on the in vitro expansion of killer cells was assessed by depleting B cells from PBMCs by microbead separation before culture. The 21-day cultures from both B-cell- and non-B-cell-depleted PBMC showed a marked expansion of NK cells, and also of T cells, among which almost half had the NKT phenotype. Depletion of B cells before culture did not change the expansion rates of NK and NKT cells significantly. In patients with progressive B-CLL, NK cell expansion capacity was improved after fludarabine treatment when compared to samples obtained before treatment. Repeated samples of PBMCs from individual untreated patients with both indolent and progressive disease cultured under identical conditions gave similar NK cell expansion rates. Expanded killer cell populations had cytotoxic function against the NK-sensitive target K562 cell line and expressed high levels of Granzyme B. From our studies, we conclude that NK cells as well as NKT cells from the peripheral blood of B-CLL patients can be expanded, and that these cells have cytotoxic capacity.

High level of cannabinoid receptor 1, absence of regulator of G protein signalling 13 and differential expression of Cyclin D1 in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a moderately aggressive B-cell lymphoma that responds poorly to currently used therapeutic protocols. In order to identify tumour characteristics that improve the understanding of biology of MCL, analysis of oligonucleotide microarrays were used to define specific gene expression profiles. Biopsy samples of MCL cases were compared to reactive lymphoid tissue. Among genes differentially expressed in MCL were genes that are involved in the regulation of proliferation, cell signalling, adhesion and homing. Furthermore, some genes with previously unknown function, such as C11orf32, C2orf10, TBC1D9 and ABCA6 were found to be differentially expressed in MCL compared to reactive lymphoid tissue. Of special interest was the high expression of the cannabinoid receptor 1 (CB1) gene in all MCL cases analysed. These results were further confirmed at the cellular and protein level by immunocytochemical staining and immunoblotting of MCL cells. Furthermore, there was a reduced expression of a regulator of G protein signalling, RGS13 in all MCLs, with a complete absence in the majority of cases while present in control lymphoid tissue. These results were further confirmed by PCR. Sequencing of the RGS13 gene revealed changes suggesting polymorphisms, indicating that downregulation of the expression of RGS13 is not related to mutations, but may serve as a new specific marker for MCL. Moreover, comparison between individual cases of MCL, revealed that the CCND1 gene appears to be differently expressed in MCL cases with high vs low proliferative activity.

Granulocyte colony-stimulating factor inhibits Fas-triggered apoptosis in bone marrow cells isolated from patients with refractory anemia with ringed sideroblasts.

Treatment with granulocyte colony-stimulating factor (G-CSF) plus erythropoietin may synergistically improve hemoglobin levels and reduce bone marrow apoptosis in patients with refractory anemia with ringed sideroblasts (RARS). Fas-induced caspase activity is increased in RARS bone marrow cells. We showed that G-CSF significantly reduced Fas-mediated caspase-8 and caspase-3-like activity and the degree of nuclear apoptotic changes in bone marrow from nine RARS patients. A decrease in mitochondrial membrane potential and an increase in intracellular reactive oxygen species occurred in Fas-treated cells, but became significant only 24 h after changes in caspase activity and decrease in proliferation. G-CSF also reduced the magnitude of these late apoptotic changes. In CD34-selected normal cells, G-CSF induced myeloid colony growth, and an overall small decrease in the number of erythroid colonies. By contrast, G-CSF induced a 33-263% increase of erythroid colony formation in CD34+ cells from four of five RARS patients with severely reduced erythroid growth, while the normal or slightly reduced erythroid growth of three other patients was not influenced by G-CSF. This study suggests that G-CSF may reduce the pathologically increased caspase activity and concomitant apoptotic changes, and promote erythroid growth and differentiation of stem cells from RARS patients. Our data support the clinical benefit of G-CSF in this subgroup of myelodysplastic syndromes.

Immunopathology of metastases in patients of colorectal carcinoma treated with monoclonal antibody 17-1A and granulocyte macrophage colony-stimulating factor.

Twenty patients with metastatic colorectal carcinoma were treated with a single infusion (400 mg) of a mouse monoclonal antibody (IgG2a) against the tumor-associated antigen CO 17-1A and with a daily injection of granulocyte macrophage colony-stimulating factor (GM-CSF) for 10 days. The cycle was repeated every month. Metastases from 5 of the 20 patients biopsied on days 1 and 10 of the first two treatment cycles were studied by immunohistochemistry. During treatment, neutrophils, monocytes, and T lymphocytes increased concordantly in the tumor as in the blood of the individual patient. Macrophages (CD68) and CD8+ T cells infiltrated the tumor glands and displayed TIA-1-reactive cytotoxic granules. Neutrophils were seen mainly in areas of necrosis. Activated (HLA-DR+) CD4+ T cells were usually abundant in the stroma. During treatment, few natural killer cells were found in the tumor, contrary to the marked increase seen in blood. Our observations indicate that GM-CSF markedly recruited activated, tumor-infiltrating leukocytes, possibly representing antibody-dependent cellular cytotoxicity and cytotoxic T effector cells. The notion that combined antibody and GM-CSF therapy may also promote a T-cell antitumor response is further supported and advocated by our findings. The study lends further support to combining GM-CSF with monoclonal antibody-based therapy.

Ex vivo T lymphocyte expansion for retroviral transduction: influence of serum-free media on variations in cell expansion rates and lymphocyte subset distribution.

OBJECTIVE: In the setting of allogeneic stem cell transplantation, suicide gene-manipulated donor T cells that can be selectively inactivated in vivo would potentially allow optimal control of the GVL (graft-vs-leukemia)/GVHD (graft-vs-host disease) balance. Retroviral T-cell transduction requires ex vivo cell expansion, which is often achieved by IL-2 and anti-CD3 stimulation. Traditionally, culture media for cell expansion are supplemented with fetal bovine serum (FBS) or human serum. While these sera promote cell growth and viability, they contain uncharacterized elements that may yield inconsistent results from batch to batch. Cell expansion in serum-free media would therefore be preferable. MATERIALS AND METHODS: We compared T-cell expansion rates in three commercially available serum-free culture media (X-VIVO 15, AIM-V, and Cellgro SCGM), with or without the addition of human serum (HS, 5%). We also aimed to evaluate how the in vitro expansion affected the composition of the various T-cell subsets. Buffy-coats from four healthy donors were expanded for 21 days. The media were compared to standard RPMI 1640 medium, supplemented with HS (5%) or FBS (10%). For retroviral transductions, the LN vector carrying the neomycin- resistance gene was used in four additional donors. RESULTS: In our hands, X-VIVO 15 gave the highest rate of serum-free expansion (a median of 79-fold expansion, range 20-117). For serum-free expansion, activation with OKT3 for 21 days gave slightly higher expansion rates than a 5-day course (however, without statistical significance). When serum was added, this discrepancy was not seen. Cytokine analysis (IFN-gamma, IL-10, and IL-4) showed a distinct type1 cytokine pattern with elevated IFN-gamma levels during the whole period of culture. Flow cytometric analyses showed substantial inter-media, but also some inter-donor, variability in T-cell subset compositions. Transduction of cells with the LN vector and G418 selection resulted in a 14-fold increase (range 3-18) for serum-free X-VIVO 15 based cultures. Cell phenotypes remained unchanged by the transduction procedure as compared to nontransduced cells. CONCLUSION: Among the tested serum-free media, X-VIVO 15 has shown to best support the in vitro expansion of T cells, resulting in equal percentages of CD4(+) and CD8(+) cells. These cells can easily be transduced and selected. There seem to be no significant benefits, regarding absolute cell numbers or T-cell subset compositions, with OKT3-stimulation for more than five days. The addition of low levels of HS increases the consistencies in the cell expansion rates for all media.

HIV prevention among injecting drug users: three years of experience from a syringe exchange program in Sweden.

Exchange of syringes and needles has for the last 3 years been offered to injecting drug users as part of an HIV prevention project in a small university town in south Sweden. The program at the local hospital has been visited by 979 drug users, of which 182 have participated on a more regular basis. The typical participant is a 30-year-old male who has injected amphetamine or heroin for at least 10 years. The seroprevalence for HIV among drug users in south Sweden has been maintained at approximately 1% in contrast to up to 60% in subpopulations from other Scandinavian regions with a comparable drug problem. No project participant has become HIV infected during the study period and a reduction in risk behavior has been noted among local drug injectors. The HIV prevention project has attracted many individuals with no previous contact with drug rehabilitation programs; for a number of drug users, the syringe exchange has served as an introduction to such treatment efforts.

Epstein-Barr virus (EBV)-encoded membrane protein LMP1 from a nasopharyngeal carcinoma is non-immunogenic in a murine model system, in contrast to a B cell-derived homologue.

Epstein-Barr virus (EBV)-encoded LMP1 gene derived from a nude mouse passaged nasopharyngeal carcinoma (NPC) of Chinese origin (C-LMP1) and its B cell (B95-8 prototype)-derived counterpart (B-LMP1) were compared for their ability to induce tumour rejection in a mouse mammary adenocarcinoma system. Each of the two LMP1 genes was introduced individually by retroviral vectors into a non-immunogenic mammary carcinoma line, S6C, that originated in an ACA (H-2f) mouse. Syngeneic ACA mice were immunised for 3 consecutive weeks with irradiated B- or C-LMP1 expressors or control cells. The immunised and control mice were then challenged with graded numbers of viable cells from the corresponding cell line. Only the B-LMP1 expressing cells were highly immunogenic. Up to 10(5) cells were rejected in pre-immunised mice, whereas at least 10(2) cells grew in non-immunised controls. No rejection response was detected against the C-LMP1 expressing cells which grew equally well in control and immunised mice, with a minimum inoculum of 10(2) cells in the majority of the clones. In a previous study, we found numerous sequence differences between B- and C-LMP1. The question of whether any of these differences is related to the non-immunogenicity of C-LMP1 needs further investigation. Meanwhile, our findings raise the possibility that the NPC cells may escape host rejection by the development of a non-immunogenic LMP1 variant under the impact of immunoselection.

Analysis of human IgA subclasses by in situ hybridization and combined in situ hybridization/immunohistochemistry.

In this report we describe a method for the analysis of the cellular expression of the two human IgA subclasses by in situ hybridization (ISH). The technique permits the detection of specific mRNA in individual cells using 35S-labeled oligonucleotide probes without any detectable cross-hybridization between the subclasses. This method was applicable both on cytospin preparation of peripheral blood mononuclear cells as well as in formalin-fixed, paraffin-embedded tissue sections. Furthermore, we describe a combined ISH/immunohistochemistry technique for the simultaneous detection of IgA subclass mRNA and protein at the single cell level. Examination of tissue sections from tonsils revealed a striking localization of labeled cells within the germinal center of some of the lymphoid follicles. The implications of this novel finding and the development of the method are discussed.

Rabbit antiserum to a citric acid extract of human skeletal muscle staining thymomas from myasthenia gravis patients.

Rabbit antiserum to a citric acid extract of human skeletal muscle (CA) stained both the cell membrane and the cross-striational bands of skeletal muscle cells. The rabbit antiserum also stained the cell membrane of epithelial thymoma cells from myasthenia gravis (MG) patients. Normal and hyperplastic thymus tissue were not stained, apart from scattered myoid thymic cells. Absorption of the antiserum with CA abolished staining of the thymoma, indicating that human skeletal muscle and epithelial thymoma cells possess common antigens.

A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells.

Adoptive transfer of immunocompetent cells may induce anti-tumor effects in vivo. However, a significant obstacle to the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Among the immunologic effector cells that are considered mediators of anti-tumor effects, those with the highest per-cell cytotoxic capacity express a natural killer (NK) cell phenotype, i.e., CD56(+)CD3(-). However, such cells are normally present only in low numbers in peripheral blood mononuclear cells (PBMCs), lymphokine activated killer (LAK), and cytokine induced killer (CIK) cell preparations. To optimize the expansion of human NK cells, PBMCs were cultured in different serum free medium supplemented with monoclonal anti-CD3 antibodies and interleukin (IL)-2 at varying concentrations. By using Cellgro stem cell growth medium supplemented with 5% human serum and IL-2 (500 U/ml) cells expanded 193-fold (median, range 21-277) after 21 days, and contained 55% (median, range 7-92) CD3(-)CD56(+) cells. The remaining cells were CD3(+) T cells, 22% (median, range 2-68) of which co-expressed CD56. The expanded cell population lysed 26 to 45% of K562 targets in a 1:1 effector to target ratio, signifying substantial cytotoxic efficacy. The described method is a simple and efficient way of expanding and enriching human NK cells. We have termed these high-yield CD3(-)CD56(+) cells cytokine-induced natural killer (CINK) cells.

N-nitrosodimethylamine and 7-methylguanine DNA adducts in tissues of rats fed Chinese salted fish.

Previous studies have demonstrated that rats fed Chinese salted fish developed carcinomas of the nasopharynx and nasal cavity. In the present work the contents of nitrosamines in salted fish from the city of Guangzhou, southern China, and the contents of nitrosamines and possible nitrosamine-induced DNA adducts in organs of rats fed the fish were analysed. Similar levels of N-nitrosodimethylamine (NDMA) were detected in tough and soft salted fish. The NDMA content in steamed fish was higher than in raw fish. In vitro incubation of salted fish with gastric juice significantly increased the level of NDMA. NDMA was found in liver and kidney from rats fed salted fish for 2 years, but no dose-dependence was found between salted fish treatment and NDMA content. The level of 7-methylguanine in rat liver DNA was found to be slightly higher than in DNA from nasopharynx. However, there were no significant differences in the level of 7-methylguanine in DNA samples from rats fed salted fish and rats fed standard diet.

High-dose chemotherapy and APSCT as a potential cure for relapsing hemolysing AILD.

Angioimmunoblastic lymphadenopathy with dysproteinemia (or dysgammaglobulinemia) (AILD) is a lymphoproliferative disorder with cytogenetic and molecular abnormalities characteristic of malignant T-cell lymphoma (angioimmunoblastic T-cell lymphoma -- AITL). We report the clinical course of a 58-year-old male patient with unusually aggressive AILD, including severe hemolysis and Guillain-Barré syndrome, who entered complete remission after CHOP therapy, but had a full relapse after 2 months. At relapse, treatment with high-dose chemotherapy followed by autologous peripheral stem cell transplantation (APSCT) with CD34 selected cells was shown to be successful. The patient is alive and disease-free 3 years after diagnosis and 32 months after APSCT. Considering the poor prognosis of the majority of patients with AILD, intensive treatment followed by APSCT, may be a subject for further studies.

Flow cytometric DNA analysis: a prognostic tool in non-Hodgkin's lymphoma.

Surgical biopsies from 234 untreated patients with non-Hodgkin's lymphoma (NHL), classified according to the Kiel nomenclature, were analysed with respect to proliferative activity (S-phase) and DNA content by flow cytofluorometric (FCF-DNA) analysis. The percentage of cells in S-phase was significantly higher in lymphomas of high compared to low grade NHL (p less than 0.001). Patients with lymphomas of high grade histology and low S-phase values (less than 5.6%) achieved complete remission (CR) more often (p less than 0.05) and survived significantly longer than those with high S-phase values (p less than 0.05). In the low grade NHL group the S-phase value did not correlate to response. S-phase correlated to survival for patients with the lymphocytic (CLL & IC) (p less than 0.05) and follicle center cell (FCC) derived (p less than 0.01) but not in blastic (LB, IB, Burkitt) NHL. DNA-aneuploidy was associated with poor response to therapy and shorter CR duration in low grade NHL (p less than 0.05 for both). However, the degree of DNA-ploidy (neardiploid or aneuploid) did not correlate to survival in any of the NHL groups analysed (high- or low grade, lymphocytic, FCC derived or blastic). The Cox regression analysis indicated that the S-phase value was a stronger predictor of survival than histopathology, stage or age, especially in low grade NHL. These results suggest that S-phase analysis should be included in the clinical evaluation of NHL patients as a prognostic indicator.

Disease-dependent changes in T-cell populations in patients with shigellosis.

In shigellosis, bacterial infection is associated with an extensive inflammation of the rectal mucosa, resulting in bloody dysentery. The role of T-cell-mediated pro-inflammatory mechanisms has been implicated in this process, but the specific role of T-cell subsets is still not well understood. In this study we attempted to identify the changes in T-cell populations in patients with shigellosis during the disease course. The T-cell subset distribution was analyzed by immunohistochemistry in the rectal mucosa and by immuno-flow cytometry in the peripheral blood. Blood and rectal biopsies were studied from patients with Shigella dysenteriae 1 (n= 11) and S. flexneri (n= 11) infection and 20 healthy age-matched controls. We found an expansion of gammadelta+T cells in the rectal mucosa, but a decrease in the percentage of gammadelta+T cells in the blood in acute shigellosis. There was also a preferential increase in CD8+ T cells in the surface epithelium of rectal tissue in patients infected with S. dysenteriae 1, but not in patients infected with S. flexneri. Our findings suggest that the rectal mucosal inflammation in shigellosis is associated with an expansion of T cells, in particular CD8+ and gammadelta+T-cell subsets in the gut mucosa, which may be of importance for the pathogenesis of shigellosis.

In vitro culture of human thymic epithelial cells in serum-free media.

Epithelial cells from human thymus were cultured in vitro at various serum concentrations and under defined serum-free conditions. A total of 238 cultures from 46 thymuses (MG and normal) were analyzed. Cells from fresh thymic tissue were explanted either as fragments or single cells after enzyme treatment. Serum-free as well as fetal calf serum (FCS) containing media based on Dulbecco's minimal essential medium and Ham's F-12 (DMEM/F-12) were found to be superior to MCDB 151 based serum-free media combinations, for the selective growth of thymic epithelial cells. In contrast, cultures based on RPMI 1640 medium supplemented with 1% FCS or more showed less epithelial cell selectivity and also supplement Ultroser G gave less fibroblast contamination. In serum-free media containing less than 0.1 mM ionic Ca, the cells had a smaller surface area and appeared more angular and also contained less keratin as compared to culture media with higher calcium contents. The development of serum-free conditions for in vitro growth of human thymic epithelial cells free of fibroblast contamination will facilitate studies of growth and maturation of the epithelial cells as well as investigations of their possible role in the development of myasthenia gravis.