In vitro tuberculin reactivity of lymphocytes from patients with tuberculous pleurisy.

Mononuclear cells in pleural fluid from patients with tuberculous pleurisy were predominantly T cells. Responsiveness of pleural fluid T cells to purified protein derivative of tuberculin were studied by the assay of cell proliferation and production of lymphocyte mitogenic factor by the stimulation with purified protein derivative. Peripheral blood lymphocytes were also studied from patients and tuberculin-positive healthy controls. The order of responsiveness was as follows: pleural fluid lymphocytes greater than peripheral blood lymphocytes of patients without effusion = peripheral blood lymphocytes of healthy controls greater than peripheral blood lymphocytes of patients with effusion. The poor response of peripheral blood lymphocytes from pleurisy patients were recovered by the elimination of adherent cells in peripheral blood lymphocytes to the level of the response of peripheral blood lymphocytes from healthy controls. T cells purified from pleural fluid mononuclear cells responded more than those from peripheral blood. These results suggested that in the pleurisy patients purified protein derivative-reactive T cells in peripheral blood did not decrease in activity, but were depressed by suppressor cells, and further suggested that highly purified protein derivative-reactive T cells were accumulated in the pleural fluid.     

In vitro synthesis of IgM rheumatoid factor by lymphocytes from patients with essential mixed cryoglobulinemia.

Peripheral blood mononuclear cells (PBMC) from patients with Essential Mixed Cryoglobulinemia (EMC) were studied for their ability to synthesize polyclonal IgM and rheumatoid factor (RF) IgM in vitro. Our results indicate: that EMC-PBMC produce smaller amounts of polyclonal IgM but higher quantities of IgM-RF than normal PBMC after pokeweed mitogen (PWM) or Staphylococcus aureus activation, so that the IgM-RF to total IgM ratio is significantly greater in EMC than in normal cultures; that enriched EMC-B lymphocytes display a significantly higher spontaneous synthesis of IgM-RF than normal B lymphocytes and that the IgM-RF B cell clones are receptive to T cell regulation. Taken together these findings suggest an expansion of B cell clones committed to IgM RF production and the presence in peripheral blood of differentiated B lymphocytes capable of secreting IgM-RF in EMC.     

In vitro stimulation of lymphocytes from patients with rheumatoid arthritis

Peripheral blood lymphocytes from patients with rheumatoid arthritis (RA) and from normal controls were compared in 20 microliters droplet cultures following stimulation with phytohemagglutinin or concanavalin A. The dynamics of proliferation were significantly changed in RA. Higher numbers of cells in culture were needed to achieve the same response. This may explain the low proliferative responses of lymphocytes from some patients with RA, and apparent changes of in vitro suppressor effects, reported by other authors. Diurnal variations of lymphocytes in RA patients were also studied. No differences in the response to mitogen of lymphocytes taken at 7 AM and 7 PM were found.     

In vitro cytotoxicity of lymphocytes from patients with `acquired'' and sex-linked agammaglobulinaemia

The in vitro cytotoxicity for fowl erythrocytes of circulating lymphocytes from nine patients with `acquired' and three patients with sex-linked agammaglobulinaemia was tested by release of sodium chromate (<sup>51</sup>Cr) after 42 hr of culture in the presence of phytohaemagglutinin (PHA). The lymphocyte cytotoxicity of three patients with `acquired' agammaglobulinaemia was low, but that of the other six and of the three patients with sex-linked agammaglobulinaemia were normal. All the `acquired' agammaglobulinaemic patients had markedly diminished thymidine uptake in response to PHA at 3 days, but a normal response at 7–10 days, but the three boys with sex-linked agammaglobulinaemia showed a normal response. The findings further demonstrate the heterogeneity of lymphocyte abnormalities in patients with agammaglobulinaemia.     

In vitro studies of lymphocytes from patients with plasma cell myeloma. I. Stimulation by mitogens and cytotoxic activities

Highly purified blood lymphocytes from patients with plasma cell myeloma were tested in different in vitro systems. The patients were untreated or had received a standardized 4-day treatment with melphalan and prednisolone every sixth week. They were tested 5 weeks after the last treatment to minimize the acute toxic effects of the drugs. Lymphocytes from healthy controls were included in each experiment.

Stimulation of lymphocytes was measured by incorporation of ¹⁴C-thymidine into DNA after activation with phytohaemagglutinin (PHA), concancavalin A (Con A) and pokeweed mitogen (PWM). Their cytotoxicity was tested against Chang cells (human cell line) and chicken red blood cells in presence of PHA or heat-inactivated rabbit antibodies to target cell antigens. Lysis was quantitated as release of radio-activity from target cells labelled with ⁵¹Cr-chromate.

Antibody-induced cytotoxicity of lymphocytes from untreated patients was normal or slightly elevated while that of treated patients was severely depressed. Also, lymphocytes from treated patients were significantly less stimulated to DNA synthesis by PWM than were control lymphocytes. PHA-induced cytotoxicity and stimulation of lymphocytes by Con A or PHA were normal in all groups.

These results suggest that treatment of myeloma patients with melphalan and cortisone selectively impairs lymphocytes which respond to PWM by DNA synthesis and which participate in antibody-mediated cytolysis.

     

In vitro activity of cefpodoxime against bacterial isolates obtained from patients with cancer

The in vitro activity of cefpodoxime, an oral cephalosporin ester, against 792 bacterial isolates representing 36 species was evaluated in comparison to that of ciprofloxacin and trimethoprim/sulfamethoxazole (TMP/SMX). Cefpodoxime inhibited the majority ofStreptococcus spp.,Haemophilus influenzae andProteus mirabilis at a concentration of 0.12 µg/ml. It was also active againstCitrobacter diversus, Escherichia coli, Klebsiella spp.,Proteus vulgaris, Serratia marcescens and methicillin-susceptibleStaphylococcus aureus isolates. Overall, cefpodoxime appeared to be less active than ciprofloxacin and TMP/SMX against many pathogens common in cancer patients.     

In vitro IgE formation by peripheral blood lymphocytes from normal individuals and patients with allergic bronchopulmonary aspergillosis.

Peripheral blood lymphocytes from five normal donors and five individuals with allergic bronchopulmonary aspergillosis (ABPA) were used for measurement of in vitro IgE formation in tissue culture preparations. IgE was measured by a sensitive radioimmunoassay using concentrated tissue culture medium (TCM) and expressed as ng IgE/ml of TCM. The results demonstrated that IgE was formed by unstimulated lymphocytes from one of five normal donors (range 1.5-2.4 ng/ml) and in two of five ABPA donors (range 2-4 ng/ml). However, when different dilutions of pokeweed mitogen were added to the tissue culture, three of five normal donors and three of five ABPA donors in remission showed enhanced IgE formation. At the time of an ABPA exacerbation in one individual, there was a definite increse in IgE synthesis up to 40 ng/ml which was suppressed by pokeweed mitogen to 17.3 ng/ml. Clear differences between normals and ABPA patients in remission are not apparent.     

In vitro reactivity of human lymphocytes in chronic uraemia: analysis and interpretation

Altered cellular immunity complicating chronic uraemia includes lymphocytopenia, thymic atrophy, impaired allograft rejection and delayed hypersensitivity in skin tests, diminished appearances of lymphocytes on skin windows, and shortened in vitro survival of uraemic lymphocytes. Studies were undertaken to further characterize these lymphocyte defects.

Lymphocytes separated from peripheral blood of twenty-four uraemic patients were compared with those from fifty-one normal persons in their rates of RNA and DNA synthesis in both PHA-stimulated and unstimulated cultures, as determined by incorporation of radiolabelled precursors. Synthesis, expressed as absolute incorporation/10⁶ viable lymphocytes, was accelerated in the majority of both stimulated and unstimulated uraemic cultures. Serial studies over several months of twenty-five uraemic patients (fifteen maintained by haemodialysis, ten by renal allotransplantation), compared with nineteen normal controls, showed these differences to be consistent and persistent. While normal lymphocytes exhibited stability, uraemic cells fluctuated widely in their serial synthesis rates. In 25% of such cultures, unstimulated synthesis exceeded that induced by PHA. Increased synthesis without PHA, reflecting enhanced spontaneous blastogenesis, is compatible with decreased survival, numbers, and functional capacity of uraemic lymphocytes.

Reports by others of diminished PHA-responsiveness of uraemic lymphocytes are based upon whole-culture incorporation and/or expressed as ratios between stimulated and unstimulated cultures. Both shortened survival and accelerated spontaneous nucleic acid synthesis by uraemic lymphocytes cause such ratios to be misleadingly low. Such factors as cell numbers and viability at harvest, counting efficiency, and culture sterility are essential to avoid misinterpretations of such data based on incorporation of radiolabelled precursors to the nucleic acids.

     

Recruitment of effector lymphocytes by initiator lymphocytes. In vivo migration of in vitro sensitized initiator T lymphocytes.

We previously found that mouse T lymphocytes sensitized in vitro against allo- or syngeneic fibroblasts, upon injection into syngeneic recipients, do not themselves differentiate into effector cells, but recruit effector T lymphocytes within the draining lymph nodes. As a result of sensitization, these initiator lymphocytes acquire a trypsin-sensitive membrane property which is necessary for recruitment. We now report studies on the in vivo migratory behavior of initiator lymphocytes following sensitization. We injected 51Cr-labeled initiator lymphocytes into recipient footpads and found significantly increased migration of sensitized cells to the draining popliteal lymph node (PLN) during the first day. By amputation of the foot at various times, we showed that migration during the first 12-24 hours was critical for subsequent recruitment. Trypsin treatment of initiator lymphocytes abolished this accelerated migration. Lymphocytes triggered nonspecifically by Con A migrated to the PLN like antigen-sensitized cells. We also compared the migration of injected lymphocytes from the footpad to the PLN in graft-versus-host and host-versus-graft reactions, and found these reactions to differ both from each other and from recruitment in terms of lymphocyte migration. These findings are discussed in terms of the physiology of the cell-mediated immune response and the notion of peripheral sensitization.     

In vitro reaction to orthopaedic biomaterials by macrophages and lymphocytes isolated from patients undergoing revision surgery

Periprosthetic tissues observed at sites of loose total joint implants exhibit abundant macrophages, lymphocytes, fibroblasts and particulate debris. Macrophages phagocytose orthopaedic debris and release proinflammatory cytokines, chemokines, matrix metalloproteinases and other substances. In addition, other cell types present in tissues harvested from the bone-implant interface are thought to influence periprosthetic bone resorption. The present study examined the effects of polymethylmethacrylate (PMMA), cobalt chrome molybdenum alloy (CoCr), and titanium-alloy particle challenge on macrophages co-cultured with lymphocytes in vitro. Potential synergistic effects of lymphocytes on macrophage activation were determined by measuring interleukin-6 and tumor necrosis factor-alpha release following exposure to orthopaedic biomaterial particles. Exposure of macrophages or macrophages co-cultured with lymphocytes to all three types of particles resulted in increased release of interleukin-6 and tumor necrosis factor-alpha at 48 h, when compared to macrophages or macrophages co-cultured with lymphocytes, respectively, cultured in the absence of particles. Lymphocytes isolated from periprosthetic tissues secreted increased basal levels of cytokines relative to peripheral blood lymphocytes. Higher doses of PMMA and titanium-alloy particles stimulated increased levels of cytokine release in the macrophage and macrophage/lymphocyte groups. In contrast, a higher dose of CoCr particles (0.075% v/v) was not as effective as the 0.015% v/v dose, indicating probable CoCr toxicity. The macrophage/lymphocyte co-culture did not show synergism between the two types of cells with respect to cytokine release. T-cells at the bone-implant interface may alter the biological response to particulate debris.     

Recruitment of effector lymphocytes by initiator lymphocytes. In vivo migration of in vitro sensitized initiator T lymphocytes

We previously found that mouse T lymphocytes sensitized in vitro against allo- or syngeneic fibroblasts, upon injection into syngeneic recipients, do not themselves differentiate into effector cells, but recruit effector T lymphocytes within the draining lymph nodes. Asaresult of sensitization, these initiator lymphocytes acquire a trypsin-sensitive membrane property which is necessary for recruitment. We now report studies on the in vivo migratory behavior of initiator lymphocytes following sensitization. We injected ⁵¹Cr-labeled initiator lymphocytes into recipient footpads and found significantly increased migration of sensitized cells to the draining popliteal lymph node (PLN) during the first day. By amputation ofthe foot at various times, we showed that migration during the first 12—24 hours was critical for subsequent recruitment. Trypsin treatment of initiator lymphocytes abolished this accelerated migration. Lymphocytes triggered nonspecifically by Con A migrated to the PLN like antigen-sensitized cells. We also compared the migration of injected lymphocytes from the footpad to the PLN in graftversus-host and host-versus-graft reactions, and found these reactions to differ both from each other and from recruitment in terms of lymphocyte migration. These findings are discussed in terms of the physiology of the cell-mediated immune response and the notion of peripheral sensitization.     

In vitro activation of lymphocytes from nonsmall cell cancer patients by interleukin 2 and anti-CD3 antibody

The current interest in adoptive immunotherapy of cancer has stimulated research into novel approaches of activating lymphocytes in vitro. We have studied the effect of anti-CD3 antibody on the in vitro activation of peripheral blood lymphocytes and tumor infiltrating lymphocytes (TIL) taken from patients with nonsmall cell cancer of the lung (NSCC). We demonstrate that anti-CD3 substantially enhances the proliferative response and bulk culture growth of interleukin 2 (IL-2)-activated killer cells. The addition of anti-CD3 to IL-2-treated TIL enhances their cytotoxicity against fresh autologous NSCC tumor targets, but not against the cancer cell lines K562 and M14. The effectors generated by culture in IL-2 and anti-CD3 have greatly increased IL-2 receptor expression and are predominantly CD4+ cells. These results establish anti-CD3 as a potentially powerful agent in the in vitro activation of lymphocytes from cancer patients.     

Thymus lymphocytes in uraemic rats and the effect of thymosin fraction 5 in vitro.

Cellular immunity of thymus lymphocytes in uraemic rats was studied. Severe and moderate uraemia was induced in rats, and sham-operated and normal rats were used as the controls. As a result, the response of thymus lymphocytes to concanavalin A (Con A) significantly decreased in severely uraemic rats, but did not change in moderately uraemic rats. However, when the thymus lymphocytes were pretreated with thymosin fraction 5, the response to Con A was ameliorated in severely uraemic rats. There was a significant correlation between the effect of thymosin fraction 5 on Con A response and Con A response of thymus lymphocytes. In addition, serum from severe uraemic rats suppressed the response of normal thymus lymphocytes to Con A. These results indicate that severe uraemia may cause an impairment in maturation of thymus lymphocytes, which can be improved by thymosin fraction 5 in vitro.     

Inhibition of the in vitro outgrowth of Epstein-Barr virus-transformed lymphocytes by thymus-dependent lymphocytes from infectious mononucleosis patients.

Known numbers of thymus-dependent (T) lymphocytes, obtained by positive selection from the blood of acute infectious mononucleosis (IM) patients and from control donors, were added to target cultures of foetal mononuclear cells within 0-7 days of exposure of the target cells to one of a range of doses of Epstein-Barr (EB) virus. The subsequent outgrowth of virus-transformed foetal cells was markedly inhibited by the presence in the cultures of IM-derived T cells, whilst similar numbers of T cells prepared either from cord blood or from adult donors seronegative for EB virus had little or no inhibitory effect. Target foetal cells treated with papain to remove any viral envelope material remaining on the cell surface after infection, were just as sensitive as untreated cells to the addition of IM-derived T cells. It is concluded that the inhibition cannot be mediated through recognition either of viral envelope structures on the surface of infected cells or of the antigenically related virus-determined membrane antigen, MA, but must depend upon recognition of the lymphocyte-detected membrane antigen, LYDMA. The regularity with which IM-derived T cells block the outgrowth of virus-transformed foetal cells suggests that LYDMA consistently appears on the surface of infected foetal cells before the establishment of transformed foci, but is unlikely to be directly associated with the cells' existing histocompatibility antigens.     

Mixed lymphocyte reactivity of human lymphocytes primed in vitro. I. Secondary response to allogenic lymphocytes.

In order to study the mixed lymphocyte culture reactivity of human lymphocytes primed in vitro, a nucleopore culture chamber technique allowing human lymphocytes to be cultured for a period of at least two weeks has been developed. During the primary culture period in nucleopore chambers, human lymphocytes were sensitized against mitomycin-treated allogenic stimulating cells. It was shown that the stimulated lymphocytes underwent a blastogenic reaction and the results suggest a reversion to the state of small, resting, primed lymphocytes. In vitro primed lymphocytes displayed allogenic memory. This was characteristic of a secondary response, which is shown by the following: 1) acceleration, the peak of thymidine incorporation occurring on day 4,2) specificity, the accelerated response was observed only when the primed lymphocytes were confronted with the cell used for priming. Contact with a third party cell did not produce this kind of activation. 3) Amplitude; the peak DNA synthesis response was greater than that of unprimed lymphocytes cultivated for the same length of time.     

Intracellular immunoglobulin production in vitro by lymphocytes from patients with hypogammaglobulinaemia and their effect on normal lymphocytes.

The ability of peripheral blood lymphocytes from twenty-two patients with late onset (acquired or common variable) hypogammaglobulinaemia to produce immunoglobulin was assessed by the immunofluorescent detection of intracytoplasmic immunoglobulin (Ic-Ig) in cultures stimulated with pokeweed mitogen (PWM). Intracellular immunoglobulin was found in 4-9-26% of cultured cells from eighteen out of nineteen controls. In contrast nineteen out of twenty-two patients with hypogammaglobulinaemia showed values less than 1% and in ten no Ic-Ig was detected. Two of the remaining three patients showed normal values. Lymphocytes from eleven patients showing less than 1% positive cells were selected for mixture experiments. Lymphocytes from five of the eleven patients strongly depressed immunoglobulin synthesis by normal lymphocytes when mixed together in the presence of PWM. However, lymphocytes from these individual patients did not depress immunoglobulin production in all normal controls.     

In vitro detection of ovalbumin-specific IgA producing cells from lymphocytes of patients with hen-egg allergy

We studied antigen-specific IgA production from the peripheral lymphocytes of children with hen-egg allergy by using the method of indirect plaque forming cell (PFC) assay. The number of OVA-specific IgA-PFC generated from the patient lymphocytes was low (6 +/- 3/7 X 10(4) non T cells) in comparison with age-matched normal children (112 +/- 18/7 X 10(4) non T cells). The numbers of OVA-specific IgG-PFC and IgM-PFC generated from the patient lymphocytes were not so different from those of the age-matched normal children. This indicates that the activity of OVA-specific IgA-antibody production was reduced in patients with hen-egg allergy.