Stimulation of Normal Lymphocytes with Autologous Lymphoid Cell Lines: Properties of Derived Killer Cells

Lymphocytes from normal adults, with or without serological signs of previous Epstein-Barr virus (EBV) infection, could be stimulated to proliferate and produce killer cells by incubation with autologous EBV-genome-positive lymphoid cell lines (LCLs). The stimulated cells were most probably of T-cell origin, although at the peak of stimulation many of them lacked the sheep erythrocyte marker. Direct effector-target cell contact was necessary for lysis to occur The cytotoxicity of autologously stimulated (AS) lymphocytes was not restricted to EBV-genome-positive LCLs, nor to cell lines of hematopoietic origin It was equally broad if cells carrying complement receptor had been removed before stimulation Fresh lymphocytes, blasts induced by phytohemagglutinin or concanavalin A. and Burkitt's lymphoma biopsy cells were resistant or considerably less sensitive Mouse cells-even cell lines-were resistant The sensitivity of target cells to lysis correlated positively with their capacity to block AS lymphocyte lysis of autologous LCLs in competition experiments The cytotoxicity of AS lymphocytes was blocked by EBV-genome-positive and-negative cell lines, whereas the EBV-related cytotoxicity of T cells from acute cases of infectious mononucleosis was blocked by EBV-genome-positive LCL only.     

Modulation of Gut-Associated Lymphoid Tissue Functions with Genetically Modified Lactococcus lactis

Lactic acid bacteria are a group of taxonomically diverse, Gram-positive food-grade bacteria that have been safely consumed throughout history. The lactic acid bacterium Lactococcus lactis, well-known for its use in the manufacture of cheese, can be genetically engineered and orally formulated to deliver therapeutic proteins in the gastrointestinal tract. This review focuses on the genetic engineering of Lactococcus lactis to secrete high-quality, correctly processed bioactive molecules derived from a eukaryotic background. The therapeutic applications of these genetically modified strains are discussed, with special regards to immunomodulation.     

Characterization of Human Lymphocytes Bearing Fc Receptors for IgE Isolated from Blood and Lymphoid Organs

Human lymphocytes isolated from adult peripheral blood and cord blood, tonsils, adenoids and spleens were analysed for Fc receptors for IgE (Fcepsilon) by rosette assays. The Fcepsilon+ cells were also characterized for their class of surface immunoglobulin (sIg), complement receptors, receptors for sheep erythrocytes (E), and Helix pomatia A haemagglutinin (HP), and their abilities to phagocytoze and adhere. The average number of Fcepsilon+ cells was in adult peripheral blood 1.2 +/- 0.4%, in cord blood 3.0 +/- 1.3%, in tonsils 4.2 +/- 5.2%, in adenoids 5.8 +/- 4.2%, and in spleens varied from 0.8% to 15.8% among individual patients. Overnight culturing of the lymphocytes under conditions that allowed detection of Fc receptors for IgM (Fcmu) usually lowered the number of Fcepsilon+ cells. Neuraminidase treatment caused no change. Rosette formation was inhibited by IgE myeloma proteins and their Fc fragments, but not by mildly reduced and alkylated and heated (56 degrees C) IgE, indicating that the receptors are specific for the native configuration of the IgE Fc fragment. Double cell surface marker analyses with fluoresceinated F(ab')2 fragments of purified anti-mu, delta, and gamma antibodies used to label the Fcepsilon rosette-forming lymphocytes from peripheral adult and cord blood showed that 50-80% were sIgM+ but only 0-28% were sIgD+. In contrast, approximately 80% of the Fcepsilon+ cells from tonsils, adenoids and spleens were sIgM+ and sIgD+. The Fcesilon+ lymphocytes represented 10-20% of the sIgM+ lymphocytes in both the peripheral adult and cord blood. Depletion and enrichment experiments indicated that most of the Fcepsilon+ cells bear complement receptors. Lymphocytes having both E and Fcepsilon receptors were not found. Furthermore, the lymphocytes with HP receptors, a marker for T cells and immature B cells, were Fcepsilon-. Monocytes and neutrophil granulocytes did not form Fcepsilon rosettes. These data indicated that a minor population of human B lymphocytes have Fcepsilon receptors. The majority of the Fcepsilon+ lymphocytes in the blood differ from those in tonsils, adenoids and spleen in that the majority of the former are sIgM+/sIgD- and the latter sIgM+/sIgD+. The sIgM+/sIgD- Fcepsilon+ cells in the blood are probably relatively mature lymphocytes since they lacked HP receptors, which are found on immature B cells.     

Immunophenotypical Characteristics of Permanent Cultures of Lymphoid Cells from Papio Hamadryas and Macaca Arctoides

Immunophenotypical characteristics of primate cells were studied by enzyme immunoassay and flow cytofluorometry using a panel of monoclonal antibodies to human B- and T-lymphocyte antigens. Specific features of immunophenotype of cultured cells were detected. Simian lymphoid cultures consist of a mixture of B- and T-cells with mosaic antigenic structure expressing markers of B and T cell specificity.     

An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible to Mycobacterium tuberculosis

I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10³ or 10⁵ CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44⁻ CD45RB⁺ cells disappeared within 2 weeks postinfection, while the number of CD44⁺ CD45RB^−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3⁺ CD4⁺ CD8⁻ T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ⁺ IL-4⁻) clone and one Th0-like (IFN-γ⁺ IL-4⁺ IL-10⁺) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.     

IgG on Infants'' B Lymphocytes: Enhanced Binding of IgG by IgM-Bearing Lymphoid Cells in Early Childhood

IgM/IgD-bearing lymphocytes (B cells) from children in the first few weeks of life were found to also have surface IgG, unlike most normal adult B cells. The IgG was loosely bound to the lymphocyte surface and was partially or completely removed by incubation at 37 degrees C or by trypsinization. When F(ab')2 antisera were employed, very few infant B cells had surface IgG, although the IgM staining was similar to that obtained with the native antisera. IgM/IgD-positive cells bound IgG anti-Rh-coated (Ripley) erythrocytes, unlike most adult B lymphocytes. Capping experiments suggested that an Fc receptor on the cells could be redistributed by the anti-IgM-surface IgM complex. These data indicate that, during infancy, B-lymphocyte receptors for IgG are of altered affinity, frequency, or availability compared with adult B-lymphocyte Fc receptors and resemble the Fc receptors found on "third population" (Fc + Ig-) mononuclear cells and monocytes.     

Lymphoid organ development: from ontogeny to neogenesis

The development of lymphoid organs can be viewed as a continuum. At one end are the 'canonical' secondary lymphoid organs, including lymph nodes and spleen; at the other end are 'ectopic' or tertiary lymphoid organs, which are cellular accumulations arising during chronic inflammation by the process of lymphoid neogenesis. Secondary lymphoid organs are genetically 'preprogrammed' and 'prepatterned' during ontogeny, whereas tertiary lymphoid organs arise under environmental influences and are not restricted to specific developmental 'windows' or anatomic locations. Between these two boundaries are other types of lymphoid tissues that are less developmentally but more environmentally regulated, such as Peyer's patches, nasal-associated lymphoid tissue, bronchial-associated lymphoid tissue and inducible bronchial-associated lymphoid tissue. Their regulation, functions and potential effects are discussed here.     

T and B Cells in Various Lymphoid Tissues in Mice: Membrane Immunofluorescence with Purified Lymphoid Cells

The proportion of T and B cells in several lymphoid tissues of the mouse was determined by membrane immunofluorescence. Lymphoid cell preparations were purified by glass-wool column and Hypoque-Ficoll sedimentation to eliminate the large majority of non-lymphoid cells which might introduce counting errors on fluorescence microscopy. FITC-labelled horse anti-mouse-globulin did not stain thymocytes (T cells) whereas it did stain a proportion of the lymphocytes of other lymphoid tissues (B cells): peripheral blood, 11%; bone marrow, lymph node, 23%; spleen, 24%. These results correlated well with the proportion of cells stained by anti-B cell sera obtained by the complete absorption of anti-lymphocyte serum (ALS) with thymocytes.     

Cytotoxic Effector Cells from Infectious Mononucleosis Patients in the Acute Phase Do Not Specifically Kill Epstein-Barr Virus Genome-Carrying Lymphoid Cell Lines

We describe a study in which we investigated the cytotoxic activities of thymusderived (T) lymphocytes and natural killer cells against Epstein-Barr virus (EBV) genome-carrying lymphoid cell lines. Purified subpopulations of lymphocytes from eight patients with infectious mononucleosis and six healthy normal EBV-seropositive donors were tested. Enriched T-cells were obtained by passing purified whole blood lymphocyte preparations through human immunoglobulin-anti-immunoglobulin-coated glass bead columns. The cytolytic activity of effector cells was determined by the ability of these cells to lyse human target cells that were internally labeled with (51)Cr. These targets included cells from both EBV genome-carrying and EBV genome-negative lymphoid lines derived from malignant tumors, as well as from lymphocytes transformed in vitro by EBV, and were chosen to represent a wide spectrum of EBV-associated membrane antigens. We found that cytotoxic T-cells from patients with infectious mononucleosis showed no EBV-related specific cell killing per se, although a trend for increased killing of cell lines derived from spontaneous in vivo growing tumors, EBV genome carrying or not, was noted; however, this trend was not observed with cell lines derived from cord blood lymphocytes after EBV infection in vitro. In addition, our data suggest that natural killer cells may play an important role in controlling EBV infection in patients with infectious mononucleosis in the acute phase of the disease, particularly since T-cells (obtained after removal on immunoglobulin-anti-immunoglobulin columns of natural killer cells presumably bearing Fc receptors) were less efficient killers than whole blood lymphocytes; furthermore, lysis by whole blood lymphocytes was also greatest against cell lines derived from malignant tumors (as opposed to in vitro EBV-transformed cord blood lymphoid lines), irrespective of whether these targets were EBV genome positive or negative.     

Permanent Impairment-Free,Relapse-Free Survival: A Novel Composite Endpoint to Evaluate Long-Term Success in Allogeneic Transplantation

Permanent impairment (PI) of vital organs is one of the transplantation-related health problems affecting the quality of life and morbidity even in patients who do not develop graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT), but no data are available on PI of multiple organs. This retrospective study aimed to estimate a novel composite endpoint of PI-free, relapse-free survival (PIRFS) in 164 allo-HCT recipients. We defined PI as >26% to 30% impairment of the whole person in 6 vital organs using the whole person impairment rating. Conventional GVHD-free/relapse-free survival (GRFS) and PIRFS at 5 years were 33.8% (95% confidence interval [CI], 26.5% to 41.3%) and 40.6% (95% CI, 32.6% to 48.4%), respectively. In the whole cohort, PIRFS was higher than GRFS at any time after allo-HCT. However, PIRFS was lower than GRFS after day 397 post-transplantation in patients who underwent umbilical cord blood transplantation (UCBT). In UCBT recipients, 5-year GRFS and PIRFS were 47.6% (95% CI, 34.3% to 59.7%) and 39.2% (95% CI, 26.6% to 51.5%), respectively. The cumulative incidence of PI after 5 years was 20.9% (95% CI, 13.7% to 29.0%) in patients surviving for ≥6 months without relapse. The multivariate analysis revealed that high disease risk (hazard ratio [HR], 1.91; 95% CI, 1.26 to 2.88; P < .01) and Karnofsky Performance Status score ≤90% at transplantation (HR, 1.73; 95% CI, 1.14 to 2.63; P = .01) were correlated with the lower PIRFS, whereas UCBT (HR, 2.35; 95% CI, 1.11 to 4.99; P = .03), grade III-IV acute GVHD by day 180 (HR, 3.59; 95% CI, 1.04 to 12.4; P = .04), and thrombotic microangiopathy by day 180 (HR, 2.74; 95% CI, 1.10 to 6.87; P = .03) were significantly correlated with a higher incidence of PI. More than 20% of long-term survivors had PI. Our data suggest that PIRFS is a useful endpoint for assessing long-term transplantation success from a different perspective than has been established previously.     

Sensitization of Human Lymphocytes Against Autologous or Allogeneic Lymphoblastoid Cell Lines: Characteristics of the Reactive Cells

Normal human peripheral blood lymphocytes were sensitized to autologous or allogeneic lymphoblastoid cells in vitro. Purified T lymphocytes were found to be able to respond both by performing DNA synthesis and by functioning as killer cells. Surface marker analysis of blast-transformed lymphocytes in the in vitro cultures showed a high proportion (around 50%) of blasts lacking any surface marker attributable to b or T blasts; such 'null' blasts have previously not been found in conventional mixed leukocyte culture or after phytohemagglutinin or concanavalin A activation Since the 'null' blasts could be shown to be produced in a high percentage from originally almost pure sheep erythrocyte(SRBC) binding lymphocytes and displayed a similar killing capacity per unit cell number is SRBC-binding lymphoblasts, we consider the 'null' blast to be of T origin.     

Arming of Macrophages with Lymphocytes from Mice Immunized with Tumor Tissue

C57BL/6 macrophages are rendered specifically cytotoxic (“armed”) by incubation with spleen cells from C57BL/6 mice sensitized in vivo with allogeneic P-815 mastocytoma cells. Data are presented which suggest that the cytotoxic activity of the macrophages is not due to the adherence of sensitized lymphocytes. The results indicate that it is the T-lymphooyte which is primarily involved in arming. Furthermore it is the cytotoxic and not the proliferating T-cell which is responsible for arming.     

地方性克汀病患者外周血淋巴细胞核T3受体研究

本文报告了利用放射配体分析方法对９例克汀病患者及１７名正常人进行了外周血淋巴细胞核Ｔ３受体的研究，通过Ｓｃａｔｃｈａｒｄ发析确定了各自的核Ｔ３受体的亲和常数和最大结合容量，结果表明，克汀病患者与正常人的亲和常数（Ｋ）一致，分别为（２．１３±０．６９）×１０＾１０Ｌ／ｍｏｌ和（１．７４±０．１３）×１０＾１０Ｌ／ｍｏｌ，而最大结合容量（ＭＢＣ）则有显著差别，分别为１８．５３±６．９７ｆｍｏｌ／１００     

Assays for Endogenous and Exogenous Lymphoid Leukosis Viruses and Chick Helper Factor with RSV(−) Cell Lines

Japanese quail cells transformed by the envelope-defective Bryan high-titer strain of Rous sarcoma virus [R(-)Q] were used as a source of the Rous sarcoma virus genome in three kinds of assays. (i) The simplest and most sensitive assay for infectious, endogenous viruses of the chicken belonging to subgroup E involved infection of a mixture of R(-)Q cells and turkey cells with the sample and assay of supernatants of these cells for focus formation on subgroup E susceptible cells. (ii) Inactivated Sendai virus-induced fusion of R(-)Q cells with live test cells was found to be a specific method for detection of chick helper factor. Focus formation by supernatant of the fused cells on subgroup E susceptible cells was correlated with the presence of subgroup E envelope glycoprotein on the plasma membranes of test cells. Whole blood cells as well as fibroblasts could be used in this assay. (iii) A method of assay for exogenous lymphoid leukosis viruses in which mixed cultures of R(-)Q cells and C/E cells and assay of supernatants for focus formation on C/E cells was as sensitive as assays presently used for exogenous lymphoid leukosis virus. Because no infectious Rous sarcoma virus was used as part of the procedure, the assays for infectious virus described here yielded pure pseudotypes of the input virus, an advantage for determining purity and subgroup of the input virus.     

Bronchopulmonary Dysplasia: Treatment Success with Dexamethasone

A newborn infant with clinical and radiologic evidence of bronchopulmonary dysplasia was successfully managed with dramatic improvement within 36 hours of treatment with dexamethasone.     

Changes in Apoptotic Gene Expression in Lymphocytes from Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients Compared with Healthy Lymphocytes

Introduction

Systemic lupus erythematosus (SLE) and rheumatoid arthritis have complex genetic traits, but in both autoimmune diseases, dysfunctional apoptosis appears to play a part in disease pathology. This study examined the levels of in vitro apoptosis in lymphocytes from healthy, rheumatoid arthritis (RA) and SLE individuals and related observed differences to their lymphocyte apoptosis gene profiles.

Materials and Methods

Lymphocytes were assessed for cell death by nuclear pyknosis and DNA fragmentation. Control, SLE and RA apoptosis gene profiles were obtained by quantitative real-time polymerase chain reaction (QRT-PCR) analysis.

Results and Discussion

The mean levels of pyknosis in RA and SLE freshly isolated lymphocytes were significantly higher than in control lymphocytes. Ninety-three apoptosis genes were analysed by QRT-PCR of mRNA from RA, SLE and healthy lymphocytes. We identified significant differences (p?<?0.05) in the expression of the same 11 of 93 and two of 93 apoptotic genes in individual SLE and RA patients tested as compared with controls.

Conclusion

We propose that similarly altered expression of specific apoptotic regulatory genes (e.g., the death effector domain-containing DNA-binding protein and apoptosis-associated speck-like protein containing a CARD) occurs in the lymphocytes of individual patients with SLE or RA that may influence the extent and rate of spontaneous apoptosis in these autoimmune conditions.     

Cytotoxic Capabilities of Lymphocytes from Patients with the Acquired Immunodeficiency Syndrome

Lymphocytes from 16 AIDS patients were tested in the cell-mediated lympholysis assay (CML). The ability to produce alloreactive cytotoxic lymphocytes in vitro was found to be substantially reduced when compared with concomitantly investigated normal controls. Addition of interleukin 2 (IL-2) to the inducer cultures increased the cytotoxic activity, but not to normal levels. The CML response did not correlate with the relative or absolute number of Leu 3+ cells or the proliferation in effector suspensions. The ability to produce cytotoxic cells in CML, and the degree of potentiation by IL-2, was positively correlated with the absolute number of Leu 2+ cells in peripheral blood of the patients, which was below normal in 56% of the patients. It is suggested that the low CML in AIDS patients is primarily caused by defective T-cell help. In addition patients with decreased absolute numbers of Leu 2+ cells may have a reduced number of CTL precursors. The natural killer (NK) activity of AIDS lymphocytes was reduced, but could be improved by incubation with IL-2 in vitro. The mononuclear cells from the patients showed a decreased ability to respond and to stimulate in the mixed lymphocyte culture. In one of the AIDS patients, the CML was found to induce autoreactivity in vitro.     

Lymphoid form of intestinal tuberculosis with miliary dissemination: case report

Tuberculosis with the incidence 28-29/100000 residents still presents a major public health problem in Croatia. Miliary tuberculosis is uncommon cause of fever of unknown origin. Intestinal tuberculosis pose as diagnostic problem that can be identified by colonoscopy and/or explorative laparatomy involving histopathology and microbiology. A case is reported of a 40-year-old HIV negative patient admitted to the Department of Infectious Diseases after two weeks of fever, diarrhea, abdominal pain and weight loss. Biochemistry testing showed mild elevation of the erythrocyte sedimentation rate and increased serum aminotransferases. On admission, chest x-ray was normal and tuberculin skin test was negative. Crohn's disease was suspected. Computed tomography of the abdomen revealed solid infiltrative mass located retroperitoneally, along with enlarged lymph nodes. Explorative laparoscopy was necessary to confirm the diagnosis. Intraoperative specimens were referred for histopathologic and microbiologic examination, which proved the existence of granulomatous inflammation of the areas with caseous necrosis. Direct microscopy of the periappendicular abscess and Ziehl-Neelsen staining of a lymph node specimen confirmed the presence of an acidoresistant bacillus. The specimen culture on solid egg based agar (L?wenstein Jensen) and liquid broth (MGIT) showed the growth of Mycobacterium tuberculosis. Then the causative agent was cultured from all specimens: sputum, stool and urine. Repeat cheast x-ray, performed on day 30 of hospitalization, showed miliary dissemination to the lungs. The patient was treated with four antituberculotics (streptomycin, isoniazide, rifampin, ethambutol) and methylprednisolone for one month, then with isoniazide, rifampin and for 11 months ethambutol. Therapy led to a decrease of abdominal lymph nodes and absence of miliary lesions on chest radiography after two months of treatment. Intestinal tuberculosis has been almost forgotten in Croatia. The latest published cases referred to HIV infected patients. In less than 50% of patients with intestinal tuberculosis the lungs are also affected, which poses a diagnostic problem. Crohn's disease is the most common diagnostic problem. Histopathology of a specimen obtained on colonoscopy and/or explorative laparoscopy can often solve the dilemma, as also confirmed in our patient. Of diagnostic studies, computed tomography has the advantage of evaluating intestinal wall involvement, which is important for the early diagnosis of intestinal tuberculosis. Enteroclysis and irrigography provide diagnostic information in the advanced stage of intestinal tuberculosis. In a patient with fever, abdominal disorders and parameters which implicate granulomatosis hepatitis or Crohn's disease, the existence of abdominal tuberculosis is also possible. Computed tomography and biopsy obtained on colonoscopy for microbiology can help in making the diagnosis and initiating appropriate treatment.