Genetic control of susceptibility to experimental Lyme arthritis is polygenic and exhibits consistent linkage to multiple loci on chromosome 5 in four independent mouse crosses.

C3H/He mice infected with Borrelia burgdorferi develop severe arthritis and are high antibody responders, while infected C57BL/6 and BALB/c mice develop mild arthritis and less robust humoral responses. Genetic analysis using composite interval mapping (CIM) on reciprocal backcross populations derived from C3H/HeN and C57BL/6N or C3H/HeJ and BALB/cAnN mice identified 12 new quantitative trait loci (QTL) linked to 10 murine Lyme disease phenotypes. These QTL reside on chromosomes 1, 2, 4, 6, 7, 9, 10, 12, 14, 15, 16, and 17. A reanalysis of an F(2) intercross between C57BL/6N and C3H/HeN mice using CIM identified two new QTL on chromosomes 4 and 15 and confirmed the location of seven previously identified loci. Two or more experimental crosses independently verified six QTL controlling phenotypes after B. burgdorferi infection. Additionally, Bb2 on chromosome 5 was reproduced in four experimental populations and was linked to the candidate locus Cora1. Evidence of four distinct QTL residing within the 30-cM region of chromosome 5 encompassing the previously mapped Bb2 and Bb3 loci was shown by CIM. Interestingly, some alleles contributing to susceptibility to Lyme arthritis were derived from C57BL/6N and BALB/cAnN mice, showing that disease-resistant strains harbor susceptibility alleles.

Children with atopic dermatitis who carry toxin-positive Staphylococcus aureus strains have an expansion of blood CD5- B lymphocytes without an increase in disease severity.

Toxin-positive strains of Staphylococcus aureus (T + S. aureus) are present on the skin of some but not all patients with atopic dermatitis. Many staphylococcal toxins are superantigens, which can stimulate the immune response and thus may potentially lead to the very high levels of IgE characteristic of this condition, as well as exacerbating the clinical disease. The aim of this study was to determine whether the presence of T + S. aureus on the skin of children with atopic dermatitis was associated with in vivo evidence of a heightened humoral immune response, higher IgE levels and more severe clinical disease. Toxin gene expression in S. aureus isolated from the eczematous lesions of 28 children with atopic dermatitis was assessed by PCR. Clinical and immune data were also collected from this cohort. Thirteen of the 28 children (46%) were colonized with T + S. aureus strains. The presence of T + S. aureus was associated with a significant expansion in peripheral blood CD5- B cells (P = 0.01), and the more toxin types identified the greater the B-cell expansion (P = 0.002). However, in this cohort of children with atopic dermatitis, despite th in vivo expansion of B cells in children harbouring T + S. aureus, there was no associated increase in IgE levels or in clinical disease severity scores.

Interleukin-4 (IL-4) and IL-13 signaling pathways do not regulate Borrelia burgdorferi-induced arthritis in mice: IgG1 is not required for host control of tissue spirochetes.

Previous studies have suggested that interleukin-4 (IL-4) has a protective effect in host defense to Borrelia burgdorferi infection, both in limiting the severity of arthritis and in controlling spirochete numbers in tissues, and a mapping study revealed suggestive linkage to a cluster of genes on mouse chromosome 11, including the genes for IL-4 and IL-13. In contrast, other studies have questioned the importance of IL-4. In this study the involvement of IL-4 in murine Lyme disease was examined in C57BL/6J and BALB/cJ mice with targeted disruptions in the IL-4 gene, the IL-4Ralpha chain gene, or both. A spectrum of arthritis severity was seen in BALB/cJ mice, and ablation of IL-4, IL-4Ralpha, or both had no effect on the overall severity of arthritis as determined by joint swelling and histopathology. Wild-type C57BL/6J mice exhibited mild to moderate arthritis, and ablation of IL-4 again had no effect on arthritis severity. IL-4- and IL-4Ralpha-deficient mice produced extremely low levels of immunoglobulin G1 (IgG1) and showed increased production of IgG2b. This shift in immunoglobulin isotype had no effect on the host's ability to control spirochete growth in either strain of mouse, as determined by PCR detection of B. burgdorferi DNA from heart and ankle tissues. In summary, the IL-4-IL-4Ralpha pathway, including IL-13 signaling, neither limits arthritis severity nor is required for control of spirochete growth during B. burgdorferi infection of mice. Furthermore, the IgG1 isotype is not required to control B. burgdorferi cell numbers in tissues. These findings suggest the host defense against B. burgdorferi infection is not dependent on the Th1-Th2 paradigm of T-cell responses.

Infection risk in patients with small cell lung cancer receiving intensive chemotherapy and recombinant human granulocyte-macrophage colony-stimulating factor.

The effect of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was assessed in 17 patients with small cell lung cancer. GM-CSF was initially given alone by subcutaneous injection for 10 days at 50-500 micrograms/m2 per day. There was a significant rise in neutrophils and eosinophils and to a lesser extent in monocytes at all dose levels. During the next phase, patients received chemotherapy (etoposide, ifosfamide and doxorubicin), and GM-CSF was given on alternate cycles, the patients acting as their own controls, so that the amelioration of chemotherapy could be assessed. Despite partial abrogation of the neutropenia associated with chemotherapy (P = 0.04), GM-CSF failed to reduce the frequency of febrile episodes in association with neutropenia, with six episodes occurring on GM-CSF and seven while patients were not receiving GM-CSF after a total of 66 cycles of chemotherapy. After GM-CSF, there was a reduction in polymorph phagocytic ability and chemotaxis in 6/12 and 9/11 patients, respectively. Timed blood counts after GM-CSF administration showed that peak leucocytosis occurred at 8-12 h and fell to two-thirds of this level at 24 h. Toxicity consisting of lethargy, myalgia and bone pain occurred at all dose levels but was manageable. 2 patients had thromboembolism. This study failed to demonstrate a reduction in the infection risk associated with moderately intensive chemotherapy for small cell lung cancer despite the partial abrogation of neutropenia.

An open study of interferon in HIV-antibody-positive men.

Alpha-2a-recombinant interferon (Roferon A) was given subcutaneously in a dose of 3 mega units twice weekly for 15-18 months to 14 HIV-antibody-positive, p24-antigen-negative men with minimal HIV-related disease. Interferon was well-tolerated and safe. Although there was either improvement or lack of deterioration initially in 22 out of 26 HIV disease markers, including lymphadenopathy, thrombocytopenic purpura and nail fungal infection, there were 11 instances of HIV disease indicators appearing during the study. At 15 months, six patients were withdrawn from the study because of clinical and immunological deterioration.

In vitro and in vivo analysis of the effects of recombinant human granulocyte colony-stimulating factor in patients.

Twelve patients with small cell lung cancer were treated with recombinant human granulocyte colony-stimulating factor, rhG-CSF, given by continuous infusion at doses ranging from 1 to 40 micrograms kg-1 day-1. Patients received the rhG-CSF before the start of intensive chemotherapy and after alternate cycles of chemotherapy. Several in vitro assays were performed using peripheral blood neutrophils and marrow progenitor cells collected from patients prior to and after infusion of the growth factor. Peripheral blood neutrophils were tested for mobility and phagocytic activity. In addition, in vitro clonogenic assays of marrow haemopoietic progenitor cells and analysis of bone marrow trephines and aspirates were carried out. We found that rhG-CSF in vivo has at least two main effects: (a) an early fall in peripheral neutrophils, within the first hour, followed by a rapid influx of mature neutrophils into the circulatory pool; (b) stimulation of proliferation and differentiation of neutrophil precursors in the bone marrow. Neutrophils released into the circulation were normal in tests of their mobility and phagocytic activity.

Proliferative response of lymphocyte subpopulations differing in avidity for sheep red blood cells after stimulation with normal allogeneic and tumour cells.

Lymphocytes from normal donors were cultured with cell lines of erythroid (K562) and lymphoid (Bri8) origin, fresh cells derived from chronic myeloid leukaemia patients (FLC), T-depleted normal peripheral blood lymphocytes (PBL) and normal bone marrow cells (NBMC). The proliferative response to these stimulator cells was evaluated in both the total lymphocyte population and in subpopulations with different affinity receptors for sheep red blood cells (SRBC), on the second, fourth and sixth days of culture. No DNA synthesis was induced by the K562 cell line, while Bri8 cells induced a proliferative response greater than that observed against PBL. No differences in the DNA synthesis pattern were observed when NBMC and FLC were used as stimulators which was comparable with that for PBL. There was a correlation between the degree of activation of the culture and the changes in size of the responding SRBC binding cell population (ET+). A progressive increase with duration of the cultures in the number of SRBC high avidity cells (EH+) and a parallel contraction of the SRBC low avidity (EL+) and E- cell populations was generally observed, so that EH+ and ET+ fractions gave similar values at the later time points. The analysis of [3H]-thymidine uptake of E+ and E- fractions on days 3 and 6, in FLC and NBMC-stimulated lymphocytes showed a prevalent involvement of E- cells on day 3, while on day 6 the majority of dividing cells belonged to the E+ compartment. A higher recruitment of E- cells in the early phase of the response was observed in FLC-stimulated lymphocytes. The participation of non-T cell populations in the proliferative response against allogeneic normal and tumour antigens is discussed.

In vitro augmentation of human natural cytotoxic activity.

Stimulation of human blood lymphocyte preparations with mitomycin C-treated lymphoid cell lines produced increased levels of cytotoxicity against both NK-susceptible and NK-resistant target cell lines. The greatest effect was seen following stimulation by the B lymphocyte-derived lines, Bri8 and raji. K562 also stimulated high levels of activity while the T lymphocyte-derived lines, CCRF/CEM and MOLT 4, produced smaller increases activity was also found in PHA- and MLC-stimulated populations. Stimulation by lymphoid cell lines gave increased cytotoxic activity against all five cell lines when used as target cells and the pattern of target cell susceptibility was maintained, with K562, CCRF/CEM and MOLT 4 being more susceptible than Bri8 and Raji. No direct correlation was found between the level of cytotoxic activity and the level of 3H-thymidine uptake in stimulated effector cell populations. The B cell lines stimulated high levels of isotopic uptake, while the T cell lines gave no significant stimulation. Similarly, the level of 3H-thymidine incorporation following PHA and MLC stimulation showed no direct correlation with the level of cytotoxic activity. Stimulation of lymphocyte transformation did not appear to be necessary for the induction of cytotoxic activity, although the largest increases in cytotoxicity occurred in populations showing high isotope incorporation. No correlation was found between the target cell susceptibility of the different cell lines and their ability to stimulate cytotoxicity.

Modulation of target cell susceptibility to human natural killer cells by interferon.

The spontaneous cytolytic activity of human peripheral blood lymphocytes in a short-term 51chromium release assay was markedly enhanced by pretreatment with partially purified "Namalva" lymphoblastoid (type I) interferon (IF), provided that the target cells, five lymphoblastoid cell lines of variable natural killer (NK) sensitivity (K562, Molt 4, CCRF/CEM, Raji and Bri8), were not similarly treated with IF. When the targets were pre-exposed to comparable concentrations of IF, their susceptibility to lysis by IF-stimulated or unstimulated effectors was diminished, while exposure of both effectors and targets to IF for the duration of the cytotoxicity assay produced levels of cytotoxicity intermediate between those obtained when either effectors or targets were pretreated. The results indicate that, depending on the experimental design. IF is capable of exerting a protective effect on certain targets, a conclusion supported by competition assays wherein IF-treated cold K562 cells competed less successfully than untreated cold K562 cells for lysis of their radiolabelled counterparts by both IF-stimulated and unstimulated effectors. The data suggest that certain lymphoblastoid targets, in common with effector lymphocytes, possess receptors for IF. Some possible biological implications of the protective interaction of IF with tumour targets, which is antagonistic to that on effector cells, are discussed.

The effect of BCG stimulation on natural cytotoxicity in the rat.

The natural cytotoxic activity of lymphoid cell populations from control and BCG-stimulated rats was examined using four target cell lines, K562, CCRF/CEM, Bri8 and Mc40. In control rats, the cytotoxicity of peritoneal cells was below that of spleen cells but above that of peripheral lymph node cells. Intraperitoneal injection of BCG induced a significant dose and time-dependent augmentation of cytotoxicity by peritoneal cells from W/Not and PVG/c rats, against all four cell lines. The increased activity reached a peak on days 4 and 5 after injection and returned to control levels by day 12. Spleen and lymph node cells from stimulated rats did not show increased cytotoxicity. K562 and CCRF/CEM target cells were considerably more susceptible to killing than Bri8 and Mc40 target cells. Separation of peritoneal cells from BCG-treated rats by density-gradient centrifugation gave an interface population, enriched with mononuclear cells showing high cytotoxic activity and a pellet population enriched with polymorphs showing very low activity. Nylon-fibre column filtration gave non-adherent and adherent cytotoxic populations. Cytotoxic activity was not diminished by removing cells adhering to Sephadex G10 or cells phagocytosing carbonyl iron, suggesting that much of the activity in this system was due to non-phagocytic mononuclear cell populations.

Enhancement of human natural cell-mediated cytotoxicity by interferon.

The effect of exogenous Namalva interferon (IF) on the natural killer (NK) cell activity of human blood lymphocytes was examined against 5 target cell lines (K562, CCRF/CEM, Molt 4, Raji and Bri8) using the 51Cr-release assay. Addition of IF to the test significantly increased the cytotoxicity, though not as much as when effector cells were treated with IF before the test. Augmentation of cytotoxicity was evident after only 1 h pretreatment and was maximal by 6 h. The rate of lysis of susceptible targets by IF-treated effectors markedly exceeded that by their untreated counterparts. Separation of lymphocyte subpopulations (by SRBC-rosette sedimentation and nylon-fibre column filtration) demonstrated that the activities of IF-stimulated and unstimulated cells were similarly distributed, suggesting that the major effect of IF is enhancement of the activity of pre-existing NK cells rather than generation of new populations of effectors. Target cell lines with high and low susceptibility to NK cells showed increased cytotoxicity by IF-treated effector cells. These findings may be relevant to the current discussion of the role of NK cells in immunosurveillance against neoplasia.

Natural cytotoxic reactivity of human lymphocyte subpopulations.

The spontaneous cytotoxicity of human peripheral blood lymphocyte preparations from normal donors for K562 target cells was examined. Effector cells were separated into SRBC rosette forming cell (RFC) and non-rosette forming cell (non-RFC) fractions using optimal and suboptimal rosetting procedures. RFC and non-RFC fractions both had high cytotoxic activity irrespective of the rosetting procedure. Owing to the larger size of the RFC fraction, it contained a higher proportion of the total activity in the preparation. Nylon fibre column adherent and non-adherent fractions also both produced cytotoxicity. Nylon fibre non-adherent cells separated by SRBC separation gave a RFC fraction with low activity and a non-RFC fraction with high activity. Separation of nylon fibre adherent cells gave RFC and non-RFC fractions with high cytotoxic activity. Therefore cytotoxic cells did not form a discrete subpopulation and either occur in several lymphocyte subsets or show a variable capacity to form SRBC rosettes and adhere to nylon fibre.

Organ distribution of natural cytotoxicity in the rat.

The natural (spontaneous) cytotoxicity (NC) of cell populations from different lymphoid organs of the rat were examined using a human myeloid cell line (K562) and a rat fibrosarcoma cell line (Mc40) as target cells. Rat blood and spleen lymphoid cell populations gave high cytotoxicity against K562, while lymph node cells and bone-marrow cells gave low levels of cytotoxicity and thymus cells virtually no activity. Addition of thymus or lymph node cells to spleen effector cells did not suppress the high cytotoxicity of spleen cells. A similar organ distribution of reactivity was observed against Mc40 cells, but the levels of cytotoxicity were much lower than for K562. A strain difference was monitored in the levels of natural cytotoxicity and cell populations from inbred Wistar rats consistently gave higher activity on a cell-to-cell basis than the corresponding population from PVG/c rats. Natural cytotoxicity was not removed when spleen cell populations were depleted of cells adhering to nylon-fibre columns or plastic surfaces, or depleted of cells ingesting carbonyl iron. In agreement with other studies using human and animal lymphoid cells, the natural killer cell in this system was found to be non-adherent and non-phagocytic and its distribution did not correspond to the established organ distribution of T or B lymphocytes.

Characterisation and separation of human lymphocytes forming mouse red cell rosettes.

Rosette formation between human lymphocytes and mouse red blood cells (MRBC) was examined as a marker for B lymphocytes and as a method of B lymphocyte separation. A small proportion of lymphocytes formed spontaneous rosettes with MRBC (mean value 6%) and the number was considerably increased by pretreating the lymphocytes with neuraminidase (mean value 16%). Double marker tests demonstrated that lymphocytes forming MRBC rosette were immunoglobulin (Ig) bearing cells, with a high proportion of IgM bearing cells, but not all Ig bearing cells formed MRBC rosettes. Lymphocyte populations enriched with T or B lymphocytes, by SRBC rosette sedimentation and nylon column filtration, gave values for MRBC rosettes consistent with a subpopulation of Ig bearing cells. Separation of MRBC rosette-forming cells gave a relatively poor degree of separation and rerosetting with MRBC produced variable results.

Human mixed lymphocyte culture using separated lymphocyte populations.

The ability of human blood lymphocyte populations enriched with T or B cells to act as responder and stimulator populations in the one-way mixed lymphocyte reaction (MLR) was investigated. T- and B-cell-enriched populations were obtained by separation of rosette-forming and non rosette-forming cells and T-cell-enriched populations were also obtained by nylon-fibre column filtration. Using cells prepared by rosette sedimentation, control unseparated and T-cell-enriched populations responded well when stimulated by mitomycin C-treated unseparated cells from a second individual; and stimulation by T- and B-enriched populations generally produced some response, although the magnitude was variable. B-cell-enriched populations gave virtually no response regardless of the composition of the stimulating populations. Nylon-column-enriched T-cell populations responded to stimulation by control unseparated cells but not to T cells purified by the same procedure. T-cell enriched populations prepared by the two methods thus had different activities in the MLR despite containing similar numbers of T cells suggesting that other factors, such as the presence of small numbers of accessory cells, are important in determining the magnitude of the MLR.

The effect of adherent and phagocytic cells on human lymphocyte PHA responsiveness.

The effect of small numbers of adherent and phagocytic cells on the human peripheral blood lymphocyte response to PHA was examined by depleting these cells from lymphocyte preparations. Lymphocyte preparations obtained by centrifugation on Ficoll--Triosil, which contained on average 85% lymphocytes, responded well to PHA. Depletion of cells adhering to nylon fibre, giving a population containing on average 95% lymphocytes, resulted in a considerably reduced response. Depletion of cells that adhered to plastic or ingested iron powder to give populations containing on average 90% lymphocytes, also reduced the PHA response, but to a lesser extent. Reduction in PHA responsiveness correlated with increasing lymphocyte purity. The responsiveness of nylon-column-filtered cells could be restored by adding a small number of cells from a monocyte-rich population.

PHA stimulation of separated human lymphocyte populations.

Lymphocyte preparations from peripheral blood and tonsils were separated into populations enriched with T or B cells by formation of rosettes with SRBC and separation of the rosette-forming and non-rosette-forming populations. T cell-enriched populations were also prepared by nylon column filtration. Using these methods preparations were obtained which comprised 80--95% T or B lymphocytes as determined by E-rosette formation and surface immunoglobulin (Ig) staining. PHA responsiveness, measured by [3H]thymidine incorporation, varied between relatively wide limits and was critically dependent on the degree of separation obtained. Relatively pure B-cell populations (less than 12% T cells) from blood and tonsils gave low PHA responses while preparations from blood still containing 24--38% T cells gave responses equal to or even greater than those of unseparated controls (60--78% T cells). T cell-enriched populations (80--86% T cells) responded to an equal or greater degree than controls but more efficient separation (greater than 90% T cells) resulted in markedly reduced stimulation. There was thus no simple correlation between the degree of phytomitogen-induced transformation and the number of T cells present. It is concluded that the low response of relatively pure T-cell populations may be due to depletion of B cells or non-lymphoid cells (or both) during the separation procedures. These observations have implications for the use of PHA stimulation as a measure of T-cell activity in mixed populations such as those of human peripheral blood leucocytes.

Stimulation of syngeneic and allogeneic lymphoid cells by tumour cells in vitro.

August and Wistar rat lymph node cells were found to respond well to PHA stimulation and in mixed lymphocyte culture, as determined by an increased incorporation of 3H-thymidine. August rat lymph node cells were also stimulated by incubation with irradiated syngeneic tumour cells. Allogeneic Wistar rat lymph node cells produced a larger response to the August tumour cells. The response of syngeneic and allogeneic lymph node cells was reduced by pretreating the tumour cells with a Wistar anti-tumour serum. Pretreating the tumour cells with sera from normal or tumour bearing rats also reduced the response of syngeneic lymph node cells but did not reduce the response of allogeneic lymph node cells.