Different proliferative and survival capacity of CLL-cells in a newly established in vitro model for pseudofollicles.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B-lymphocytes that manifests in a variety of clinical courses. The accumulation of CLL-cells is primarily caused by defective apoptosis; however, a higher proliferative capacity has also been found to correlate with poorer prognostic factors. Proliferating CLL-cells are confined to specialized structures called pseudofollicles, which contain CLL-cells, T-lymphocytes, and stromal cells. We established an in vitro model for pseudofollicles to characterize the behavior of CLL-cells in relation to clinical courses with different outcomes. Only CLL-cells from progressive clinical cases were inducible to proliferate by a combination of soluble CD40L/IL-2/IL-10 in co-culture with stromal cells. Proliferating CLL-cells showed a higher and more extensive expression of antigens, which are important in T-B-cell interactions such as CD40, MHC II, and adhesion molecules. IL-4 increased interferon regulatory factor-4 expression and induced a specific immunophenotype, which may imply plasmacytic differentiation. Furthermore, it was shown that co-cultured stromal cells protected CLL-cells from apoptosis. CLL-cells from clinically indolent cases had a far worse survival rate in medium than the cells from poor prognostic cases. Thus, we can assume that not only a different resistance to apoptosis, but also proliferation contributes to the progression of CLL resulting in bone marrow failure with thrombocytopenia and anemia.

Intranasal IFNgamma extends passive IgA antibody protection of mice against Mycobacterium tuberculosis lung infection.

Intranasal inoculation of mice with monoclonal IgA against the alpha-crystallin (acr1) antigen can diminish the tuberculous infection in the lungs. As this effect has been observed only over a short-term, we investigated if it could be extended by inoculation of IFNgamma 3 days before infection, and further co-inoculations with IgA, at 2 h before and 2 and 7 days after aerosol infection with Mycobacterium tuberculosis H37Rv. This treatment reduced the lung infection at 4 weeks more than either IgA or IFNgamma alone (i.e. 17-fold, from 4.2 x 10(7) to 2.5 x 10(6) CFU, P = 0.006), accompanied also by lower granulomatous infiltration of the lungs. IFNgamma added prior to infection of mouse peritoneal macrophages with IgA-opsonized bacilli resulted in a synergistic increase of nitric oxide and TNFalpha production and a 2-3 fold decrease in bacterial counts. Our improved results suggest, that combined treatment with IFNgamma and IgA could be developed towards prophylactic treatment of AIDS patients, or as an adjunct to chemotherapy.

Time course of mycobacterial infection of dendritic cells in the lungs of intranasally infected mice.

SETTING: Dendritic cells (DC) could regulate between the protective and pathogenic immune responses following tuberculous infection. In this paper we investigated if their early infection in the lungs represents a plausible alternative to cross-priming with mycobacterial antigens acquired from infected macrophages. OBJECTIVE: To determine the extent and time course of infection of lung DCs following intranasal inoculation of BALB/c mice with green fluorescent protein (GFP) tagged Bacillus Calmette-Guerin (BCG). RESULTS: A fraction of GFP-BCG infected lung cells were classified as monocytic DCs with the CD11c+IA+33D1+CD8a- phenotype. These cells represented 5-18% of the total GFP+ cells, the bulk of which were macrophages. The infected DCs could be separated by cell size into two fractions with similar cell surface staining properties during the 2-72 h period after infection. An unexpected difference was observed for the time course of infection between DCs and macrophages: DC infection peaked at 48 h followed by decline at 72 h, while the proportion of infected macrophages remained steady during the same period. CONCLUSION: The presented results are direct evidence that monocytic DCs are recruited to the lungs and take up live bacilli within 48 h of intranasal infection with GFP-BCG. This finding is pertinent for the regulation of pulmonary and systemic immune responses and possibly for the dissemination of mycobacterial infection by DCs.

Intranasal bacille Calmette-Guerin (BCG) vaccine dosage needs balancing between protection and lung pathology.

Intranasal vaccination may offer practical benefits and better protection against respiratory infections, including tuberculosis. In this paper, we investigated the persistence of the Mycobacterium bovis-strain bacille Calmette-Guerin (BCG) Pasteur, lung granuloma formation and protection against pathogenic tuberculous challenge in mice. A pronounced BCG dose-dependent granulomatous infiltration of the lungs was observed following intranasal, but not after subcutaneous, vaccination. Corresponding doses of BCG, over a 100-fold range, imparted similar protection against H37Rv challenge when comparing the intranasal and subcutaneous vaccination routes. Interestingly, a BCG dose-dependent reduction of the H37Rv challenge infection was observed in the lungs, but not in the spleens, following both intranasal and subcutaneous vaccination. In the light of the observed concurrence between the extent of granuloma formation and the level of protection of the lungs, we conclude that intranasal vaccination leading to best protective efficacy needs to be balanced with an acceptable safety margin avoiding undue pathology in the lungs.

Cytokine profile, HLA restriction and TCR sequence analysis of human CD4+ T clones specific for an immunodominant epitope of Mycobacterium tuberculosis 16-kDa protein.

The identification of immunodominant and universal mycobacterial peptides could be applied to vaccine design and have an employment as diagnostic reagents. In this paper we have investigated the fine specificity, clonal composition and HLA class II restriction of CD4+ T cell clones specific for an immunodominant epitope spanning amino acids 91-110 of the 16-kDa protein of Mycobacterium tuberculosis. Twenty-one of the tested 28 clones had a Th1 profile, while seven clones had a Th0 profile. None of the clones had a Th2 profile. While the TCR AV gene usage of the clones was heterogeneous, a dominant TCR BV2 gene family was used by 18 of the 28 clones. The CDR3 regions of BV2+ T cell clones showed variation in lengths, but a putative common motif R-L/V-G/S-Y/W-E/D was detected in 13 of the 18 clones. Moreover, the last two to three residues of the putative CDR3 loops, encoded by conserved BJ sequences, could also play a role in peptide recognition. Antibody blockade and fine restriction analysis using HLA-DR homozygous antigen-presenting cells established that 16 of 18 BV2+ peptide-specific clones were DR restricted and two clones were DR-DQ and DR-DP restricted. Additionally, five of the 18 TCRBV2+ clones recognized peptide 91-110 in association with both parental and diverse HLA-DR molecules, indicating their promiscuous recognition pattern. The ability of peptide 91-110 to bind a wide range of HLA-DR molecules, and to stimulate a Th1-type interferon (IFN)-gamma response more readily, encourage the use of this peptide as a subunit vaccine component.

The humoral response in TCR alpha-/- mice. Can gammadelta-T cells support the humoral immune response?

An optimal humoral response requires T-cell help; however, it has been questioned if this help comes exclusively from alphabeta-T cells or whether gammadelta-T cells also contribute. We have attempted to answer this question by studying the humoral response in T-cell receptor alpha-chain knockout (alpha-/-) mice, which lack the alphabetaT cell subset. Two model antigens were used to characterize the response: the thymus-independent (TI) antigen native dextran B512 (Dx), and the thymus-dependent (TD) antigen heat shock protein (HSP65) from Mycobacterium tuberculosis. When challenged with Dx, the alpha-/- mice elicited a strong antibody response and formed rudimentary germinal centres (GCs), a T-cell dependent reaction. In contrast, the humoral response to HSP65 was poor. However, alpha-/- mice became primed when challenged with HSP65, because when supplemented with wild-type thymocytes, the antigen-primed animals were able to mount a stronger response than the nonprimed ones when challenged with HSP65. A crucial step seems to be the collaboration between gammadeltaT cells and antigen presenting cells (APCs), as splenocytes from alpha-/- mice were able to respond to HSP65 in an environment containing primed-APCs. Based on these results, we propose a model for B-cell activation in the alpha-/- mice.

Effective anti-tumor immunity induced in mice by a two-step vaccination protocol.

BACKGROUND: TS/A cells (a Balb/c-derived tumor cell line), when injected into syngenic mice, give rise to rapidly growing tumors. In this study, a vaccination protocol was established which was able to elicit an immune response effective in controlling tumor growth. MATERIALS AND METHODS: T19.2.1, a TS/A clone enginereed to stably express the mycobacterial cell wall-associated 19-kDa lipoprotein, was used as cell vaccine to immunize Mycobacterium Bovis-BCG pre-immunized Balb/c mice. RESULTS: Mice receiving the two-step vaccination protocol were able to develop a strong anti-TS/A DTH reaction. Moreover, following a challenge with wild-type TS/A cells, some vaccinated animals rejected the tumor and the remaining animals showed a significantly increased survival in respect to controls. CONCLUSION: The expression on TS/A cells of the mycobacterial 19-kDa antigen, recognised in the context of a pre-existing memory immune response, promotes the immunological recognition of the otherwise non-immunogenic wild-type TS/A cells.

Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vgamma9/Vdelta2 T lymphocytes.

Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

[Multiple lymphomatous polyposis--rare case of gastrointestinal hemorrhage]. / Multiplex lymphomatosus polyposis--gastrointestinalis vérzés ritka esete.

The authors report a case of a 57 years old male patient, who was admitted to gastroenterology department with upper gastrointestinal haemorrhage. The urgent upper gastrointestinal endoscopy revealed an ulcerated polypoid tumor in the region of angulus of the stomach, and multiple polypoid lesions in the bulbar part of the duodenum. Upon this endoscopic appearance colonoscopy was performed, which revealed a polyposis syndrome in the colorectum. Computer tomography detected mesenterial, retroperitoneal and mediastinal lymph node involvement as well. In this case the primary or secondary origin of the gastrointestinal lymphoma was not verifiable. According to literature data this histological type of the gastrointestinal lymphoma has poor response to chemotherapy, the prognosis is unfavourable. In this particular case the administered chemotherapy resulted in total remission at the lymphoma patient clinically staging III Ae. In the proper follow-up examinations of the patient upper and lower endoscopy, histology samples, laboratory parameters, computer tomography, and physical examination in every 3 months are the methods.

A method for detection of doxorubicin-induced cardiotoxicity: Flow-mediated vasodilation of the brachial artery.

BACKGROUND: The cardiotoxicity of anthracyclin antibiotics such as doxorubicin (DOX) is a serious side effect in cancer therapy. Reduced antioxidant capacity may be a factor responsible for DOX-induced oxidative damage to the heart. The endothelial dysfunction that results from excessive free radicals can be easily detected by flow-mediated vasodilation (FMD) of the brachial artery. OBJECTIVES: To determine the change in endothelial function after intravenous DOX bolus; to determine the change in biochemical parameters, reflecting increased activity of free radicals or decreased endogenous antioxidant capacity, after intravenous DOX bolus; and to determine the relation between alteration of en-dothelial function after the first DOX bolus and the change in left ventricular function during follow-up. PATIENTS AND METHODS: Twenty-two patients, with either non-Hodgkin's lymphoma (18 patients) or Hodgkin's disease (four patients) were enrolled for the study (seven women and 15 men), with a mean age of 37.3+/-13.7 years. Each patient was treated with a DOX-containing regimen. The actual mean dose of DOX was 33+/-12 mg/m(2). FMD was evaluated before and after DOX administration. In nine patients more frequent measures were taken to determine the time course of change in FMD after DOX administration. FMD was measured 6, 12, 24 and 48 h after DOX. On average each patient was followed up for 18.6+/-8.1 months following the first DOX administration. The mean cumulative DOX dose was 229+/-112 mg/m(2) by the end of the follow-up period. Left ventricular ejection fraction was determined regularly during and at the end of the study. RESULTS: FMD was normal (more than 5%) at baseline in each patient but decreased significantly after DOX bolus (9.9+/-4.4% versus 6.1+/-4.6%, P<0.02). Marked individual differences were found in FMD changes after DOX. Patients who had a more than 5% decrease in FMD after DOX bolus were pretreated with 1000 mg of vitamin C intravenously, just before the next intravenous bolus of DOX was given. The decrease in FMD was prevented. Stepwise multiple regression analysis showed that the decrease in left ventricular ejection fraction during follow-up significantly and independently correlated with the cumulative DOX dose and the value of FMD alteration after the first DOX bolus administration. CONCLUSIONS: FMD in the brachial artery was significantly impaired after the first DOX bolus. The marked individual differences suggest different antioxidant capacities in these patients. The results suggest that alterations in FMD after DOX allows for detection of patients with insufficient antioxidant capacity and patients at a higher risk of DOX-induced cardiotoxicity.

Resistance of natural killer T cell-deficient mice to systemic Shwartzman reaction.

The generalized Shwartzman reaction in mice which had been primed and challenged with lipopolysaccharide (LPS) depends on interleukin (IL)-12-induced interferon (IFN)-gamma production at the priming stage. We examined the involvement in the priming mechanism of the unique population of Valpha14 natural killer T (NKT) cells because they promptly produce IFN-gamma after IL-12 stimulation. We report here that LPS- or IL-12-primed NKT cell genetically deficient mice were found to be resistant to LPS-elicited mortality. This outcome can be attributed to the reduction of IFN-gamma production, because injection of recombinant mouse IFN-gamma, but not injection of IL-12, effectively primed the NKT cell-deficient mice. However, priming with high doses of LPS caused mortality of severe combined immunodeficiency, NKT cell-deficient, and CD1-deficient mice, indicating a major contribution of NKT cells to the Shwartzman reaction elicited by low doses of LPS, whereas at higher doses of LPS NK cells play a prominent role. These results suggest that the numerically small NKT cell population of normal mice apparently plays a mandatory role in the priming stage of the generalized Shwartzman reaction.

Change of Th0 to Th1 cell-cytokine profile following tuberculosis chemotherapy.

T cells mediate protection against tuberculosis, but little is known about their role during chemotherapy of patients with active disease. Here we examined the cytokine profile of CD4 T cells before and after four months of chemotherapy in six initial skin test anergic cases. Purified protein derivative (PPD) and 16-kDa antigen-reactive CD4 T-cell clones prior to therapy resided mostly in disease-associated body fluids and were of the Th0 (interferon (IFN)-gamma + interleukin (IL)-4) secreting profile. In contrast, the majority of postchemotherapy CD4 T-cell clones originated from blood and were of the IFN-gamma secreting Th1 type. However, the recognition of several peptides derived from the 16-kDa antigen was not significantly different between the Th1 and Th0 clones. We conclude that chemotherapy shifts CD4 T cells from the affected body fluids to the blood circulation, accompanied by a change from Th0 to Th1 cytokine profile.

Transmission of IgA and IgG monoclonal antibodies to mucosal fluids following intranasal or parenteral delivery.

BACKGROUND: The efficacy by which passive antibodies can reach the lungs could be important for the outcome of immunotherapy of respiratory pulmonary infections. We examined how transmission to a number of mucosal sites is affected by the route of inoculation. METHODS: Transmission of newly raised IgA class Mabs against mycobacterial surface antigens to saliva, lung or vaginal lavage, bile and serum of BALB/c mice was compared with existing IgG Mabs. ELISA was used for testing body fluids obtained 1-24 h after intranasal or intravenous inoculation and 1-7 days following back-pack tumour growth of hybridomas. RESULTS: Intranasal inoculation resulted in a rapid rise and high levels of both IgA and IgG class Mabs in lung lavage. In contrast, following intravenous Mab injection or back-pack tumour growth of hybridoma cells, effective lung transmission was observed for the IgG1 and IgG2b MAbs, but not for the IgA Mabs. The secretory component was acquired by the transmitted IgA MAbs in the mucosal fluids, but not in the serum. Nevertheless, the time course of mucosal IgA antibody levels was similar to that of the tested IgG Mabs. Furthermore, the relative proportion of transmission to saliva and bile varied between individual Mabs indicating a role of tissue-specific, immunoglobulin class-unrelated mechanisms. CONCLUSIONS: Intranasal, rather than parenteral inoculation of mice is required for the efficient delivery of IgA antibodies against respiratory pulmonary pathogens. Interestingly, IgA-secretory component complexing of intranasally applied Mabs did not significantly influence their persistence in the lungs.

Intranasal vaccination of mice against infection with Mycobacterium tuberculosis.

The intranasal (i.n.) route of immunisation, has recently been of active interest in endeavours to improve the efficacy of vaccination against a number of respiratory infections. Here, we examined the outcome of tuberculous infection in BALB/c mice. I.n. application of the BCG-Pasteur strain was found to be highly protective against challenge infection with the pathogenic H37Rv strain given after a 4-week interval, reflected by the 100-fold reduction of CFUs in both lungs and spleens. Vaccination with the recombinant PstS-1 antigen and cholera toxin significantly protected against the challenge given 10 days later, but only marginally after 12 weeks. Histological examination showed, that i.n. vaccination abrogated the confluent infiltration of lungs with inflammatory cells, which surrounds the granulomas in H37Rv challenged control mice. In conclusion, the strong protection demonstrated by BCG suggests that the i.n. route of vaccine delivery deserves further attention toward improving vaccination against tuberculosis.

The existence of lymphoid lineage restricted Philadelphia chromosome-positive acute lymphoblastic leukemia with heterogeneous bcr-abl rearrangement.

Analysis of lineage involvement was performed in 17 Philadelphia chromosome-positive acute lymphoblastic leukemia patients with no history of chronic myeloproliferative disorder. The percentage of blastic cells as defined by flow cytometry matched that of the Ph-positive cells in 14 out of 17 patients. The bcr-abl rearrangement was investigated by fluorescent in situ hybridization in morphologically identified blastic cells, myeloid elements, lymphocytes and erythroblasts using a combined light and fluorescent microscopical imaging. Lymphoid lineage restriction could be determined in all but three of the patients. These 14 patients exhibited heterogeneity in terms of m-bcr and M-bcr types of translocation as revealed by reverse transcription polymerase chain reaction. The three patients with multilineage involvement and M-bcr type of translocation reverted to chronic phase and the percentage of Ph-positive cells remained high. Thus, we could identify an uncommitted stem cell origin among Ph-positive ALLs only in those patients whose disease subsequently proved to be a lymphoid blastic crisis with clinically silent chronic phase.

Vgamma9/Vdelta2 T lymphocytes reduce the viability of intracellular Mycobacterium tuberculosis.

An effective immune response against the intracellular pathogen Mycobacterium tuberculosis is strictly dependent on T cell activation. Although this protective response mainly depends on local release of pro-inflammatory cytokines by Th1 CD4(+) T cells, contribution of Vgamma9 / Vdelta2 T lymphocytes to immune protection against this pathogen is suggested by the antimycobacterial reactivity of this subset and its ability to produce large amounts of Th1 cytokines. Here we show that Vgamma9 / Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis. The cytotoxic activity of Vgamma9 / Vdelta2 T lymphocytes was not MHC class I or class II restricted but was blocked by anti-TCR monoclonal antibodies, thus indicating that it involved specific interaction between the TCR and the target cell. The cytotoxicity of Vgamma9 / Vdelta2 T lymphocytes was not mediated by TNF-alpha or Fas-Fas ligand, but was shown to occur through a granule-dependent mechanism that resulted in reduction of the viability of intracellular bacilli. Perforin was shown to play an important role in killing of both infected macrophages and intracellular mycobacteria. These data strongly suggest that Vgamma9 / Vdelta2 T lymphocytes contribute to the host defense against M. tuberculosis infection.

Recessive expression of the H2A-controlled immune response phenotype depends critically on antigen dose.

Major histocompatibility complex (MHC) alleles acting as immune response genes are coexpressed in heterozygous individuals and therefore control of immune responses is usually codominant. As an exception to this rule, however, several examples of recessive immune responses have been ascribed to regulatory, e.g. suppressive, interactions. We report here that the recessive phenotype of both antibody and T-cell responses to the mycobacterial 16 000-MW antigen depends critically on a low antigen dose for immunization. On the basis of similar responses in hemi- and heterozygous mice, we suggest that the mechanism of recessive MHC control does not involve regulation by the low-responder allele. We also demonstrated mixed haplotype restriction of peptide recognition for a significant fraction of high-antigen-dose primed T cells. Their paucity under limiting antigen dose conditions may lead to the recessive expression of MHC control. In conclusion, our results suggest that recessive MHC control can be explained as a simple gene dosage effect under conditions where antigen is limiting, without a need for regulatory mechanisms.

Ligand-specific alphabeta and gammadelta T cell responses in childhood tuberculosis.

The alphabeta and gammadelta T cell responses were analyzed in the peripheral blood of children affected by active tuberculosis (TB) and in healthy children who tested positive (PPD+) or negative (PPD-) for purified protein derivative. PPD+ healthy and diseased children responded equally well to PPD in vitro. In contrast, only 18% of PPD+ TB patients responded to peptide p38G derived from the 38-kDa protein of Mycobacterium tuberculosis. Analysis of the whole gammadelta T cell population and of its Vgamma9/Vdelta2 subset showed similar frequencies in PPD+ children with TB and in healthy PPD+ and PPD- children. Vgamma9/Vdelta2 cells from children with TB responded to 5 different phosphoantigens similarly to those from healthy PPD+ children, but healthy PPD- children responded very poorly. Chemotherapy had contrasting effects on the tested lymphocyte population, represented by increase of alphabeta and decline of Vgamma9/Vdelta2 T cell responses. T cell responses in childhood TB may be similar to those in adult TB.