Monocyte response to LPS after exposure to corticosteroids and chloroquine with implications for systemic lupus erythematosus.

Essential part of a response to infection is early pathogen recognition and adequate initiation of innate immunity. One of the hallmarks of systemic lupus erythematosus (SLE) is reduced resistance to infection despite overall hyperactivity of the immune system. Immunosuppressive drugs (high-dose corticosteroids and cytotoxic agents) are independent risk factors for infection in SLE, with bacteria as predominant cause. To investigate whether less aggressive immunomodulatory treatment may still affect recognition and response to Gram-negative bacteria, we measured TLR4 expression in monocytes of untreated SLE patients and patients on chloroquine and low-dose steroid therapy and examined the drugs' influence on monocyte TLR4 expression in peripheral blood mononuclear cell (PBMC) culture. Additionally, we determined whether induction of monocyte NF-κB signalling, TNF-α and IL-6 production with lipopolysaccharide (LPS), a TLR4 ligand, can be altered with dexamethasone, chloroquine or both. There was no statistically significant difference in TLR4 expression between patients with SLE and controls, even though treated SLE patients tended to have lower frequency of TLR4(+) monocytes and TLR4 mean fluorescence intensity than healthy controls. However, neither dexamethasone nor chloroquine had major influence on TLR4 expression in vitro or suppressed LPS-induced NF-κB activation in monocytes, although dexamethasone decreased TNF-α and IL-6 production. Therefore, even if low-dose steroids or chloroquine do not seem to affect TLR4 expression and signalling, steroids might decrease cytokine production in response to LPS.

Indirect demonstration of the lifetime function of human thymus.

The aim of this study was to test the hypothesis that human thymus maintains its function as the site of early T cell development throughout life, but to a progressively diminishing extent. Mononuclear cell suspensions prepared from the samples of 39 human thymuses were analysed for the total number of cells per gram of thymus tissue, percentage of single marker-positive CD2, CD4 and CD8 cells, percentages of double-positive CD4CD8 and CD2CD8 cells, double-negative CD4CD8 cells, absolute numbers of these cells per gram of tissue, and extent of the in vitro proliferation upon stimulation with concanavalin A (Con A), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) mitogens. The main outcome measures were flow cytometric data on thymus lymphoid cell composition (according to CD classification), expressed as percentages and numbers of cells per gram of thymus tissue. The total number of mononuclear cells expressed per gram of thymus tissue exponentially decreased with age. The slope of none of the analysed cell subpopulations differed from the slope of the line constructed for age-related decline of the total number of mononuclear cells (-0.024 on a semilogarithmic scale). The thymuses of all ages contained all analysed cell subpopulations in approximately the same proportions: percentages of these cell subpopulations did not change with age, except for all CD4+ (P=0.017) and double-positive CD4+CD8+ (P=0.016) cells, which tended to decrease with age. The extent of proliferation of thymus cells upon stimulation with T and B cell mitogens was unrelated to age. We conclude that the thymus retains its function as the site of differentiation of T lymphocytes throughout life. With respect to the number of involved lymphoid cells, the function exponentially decreases with age.

Binding of anti-double stranded (ds) DNA-positive sera to denatured (d) DNA and synthetic poly[dA-dT] x poly[dA-dT] double stranded copolymer in an ELISA format.

Using an ELISA assay anti-nuclear antibody-positive sera from 300 patients with various immune-related diseases and 64 anti-nuclear antibody-negative sera were analysed for binding to S1-nuclease-treated double stranded (ds) DNA. In addition, the pattern of reactivity of 50 selected anti-dsDNA-positive sera was established using denatured (d) DNA and poly[dA-dT] X poly[dA-dT] double-stranded alternating copolymer (dAT) as additional DNA antigens. None of the 64 anti-nuclear antibody-negative sera and 76 of the 300 anti-nuclear antibody-positive sera (25%) were anti-dsDNA-positive. Of the anti-nuclear antibody-positive and anti-dsDNA-positive sera, 48 (63%) were from systemic lupus erythematosus patients, and 7 (9%) from rheumatoid arthritis patients, whereas 21 patients (27.6%) suffered from various immune and non-immune related diseases. Anti-dsDNA-positive reactivity was highly correlated with dDNA and dAT reactivity (r = 0.906, p < 0.0001 and r = 0.93, p < 0.0001, respectively). Although the majority of the 50 selected (37 systemic lupus erythematosus and 13 non-systemic lupus erythematosus) anti-dsDNA-positive sera concomitantly bound to both additional antigens, 7 of these (14%) did not bind to dAT, and 2 (4%) did not bind to dDNA. Anti-dsDNA-positive sera (n = 37) showed a similar pattern, in which 8.1% and 2.7% of sera did not bind to dAT and to dDNA, respectively. In contrast, anti-dsDNA-negative sera from various immune-related diseases bound either ssDNA (12.5%) or dDNA and dAT (12.5%). These data suggest that dsDNA and dAT-based assays detect similar but not identical specificities in the sera of patients suffering from systemic lupus erythematosus and in a proportion of non-systemic lupus erythematosus patients.

Immunocytochemical reactivity of a mouse monoclonal antibody CDI 315B raised against human breast carcinoma.

Our previous report revealed the production of monoclonal antibody (MoAb) CDI 315B by immunization of mice with tumor extract proteins of human invasive ductal breast carcinoma. In the present study we report on the immunocytochemical reactivity of this MoAb with formalin or methacarn fixed, paraffin embedded tissue sections and also with cell cultures. Among breast tissues, positive staining was detected in 88% (64 of 73) of primary breast carcinomas, 77% (7 of 9) of metastatic lymph nodes, 24% (8 of 33) of benign breast disease and 15% (2 of 13) of normal breast tissue. No immunostaining was detected with several other tumors, with the exception of melanoma, where 63% (5 of 8) of positive staining was found. On in vitro cell lines, positive reaction was detected only with breast carcinoma and melanoma cells, but not with other examined cell lines. On benign breast disease tissue sections, positive reaction was detected in areas with cell hyperplasia. On normal breast tissue sections MoAb 315B stained the epithelial cells of terminal ductuli. Since the MoAb 315B recognized some antigen present in the cytoplasm of most breast carcinoma cells, this MoAb may have potential application in diagnosis and management of breast cancer.

Helper effects required during in vivo priming for a cytolytic response to the H-Y antigen in nonresponder mice.

Induction of H-Y-specific cytotoxic T lymphocyte (CTL) responses in nonresponder female mice was attempted by i.v. injection of allogeneic male cells, followed by in vitro restimulation of recipient spleen cells with syngeneic male cells. Responses were obtained only in two strain combinations in which the recipients, although phenotypically nonresponders, carried responder alleles at class I major histocompatibility complex (MHC) loci, and the immunizing cells differed from the recipients at class II MHC loci. The two positive strain combinations were B10.A(2R) anti-B10.A(4R), and B10.GD anti-B10.D2(R101). In the first combination, both recipient and donor are nonresponders to H-Y, and the CTL are induced via a bystander effect of another CTL response to a previously undetected minor histocompatibility (H) antigen. This "carrier" antigen can only induce CTL against H-Y and itself when the immunizing cells express class II MHC molecules. Furthermore, the presence of H-Y and the carrier antigen on the same cell is a prerequisite for the generation of H-Y-specific CTL. In the second combination, the recipient is a nonresponder, whereas the donor is a responder. The two strains differ at only E alpha and E beta class II MHC loci. For the induction of CTL, H-Y and the foreign E molecule must be expressed on the same cells. Thus, the B10.D2(R101) cells that express E molecules on their surface probably provide the E-nonexpressor B10.GD recipients with a stimulus for the generation of H-Y-specific T helper cells. The data are consistent with the notion that antigen-specific class II MHC-restricted T helper cells are involved in the initiation of CTL responses to minor H antigens.

H-41, a new minor histocompatibility locus. I. Histogenetic analysis.

The B10.STA12 mouse congenic line inherited from the wild mouse parent not only the H-2w13 haplotype but also an allele at a minor H locus, which we designate H-41. This allele (H-41a) differentiates the B10.STA12 line from B10.STA10 and B10.LIB55, which carry identical H-2w13 haplotypes but a different H-41 allele (the H-41b, also present in the background strain C57BL/10Sn). The B10.STA12 and B10.STA10 lines reject each other's skin grafts and generate cytolytic T lymphocytes (CTL) after in vivo immunization and in vitro restimulation with cells of the partner strain. The B10.STA12 anti-B10.STA10 CTL react with B10.STA10, B10.LIB55, and B10.STA39 target cells and with cells of F1 hybrids between the responder strain B10.STA12 and strains C57BL/6, C57BL/10, C57L, BALB/c, A, AKR, WB, DBA/1, and DBA/2 but fail to react with (C3H x B10.STA12) F1 and (CBA x B10.STA12) F1 cells. The B10.STA10 anti-B10.STA12 CTL react with B10.STA12, B10.P, and C3H.NB cells but fail to react to (B6 x B10.STA10) F1 target cells. The CTL reactivity in both combinations is Dp restricted. The B10.STA10 anti-B10.STA12 CTL exhibit, in addition, a cross-reactivity with B10.SAA48 cells that may be directed at one of the alloantigens controlled by the H-2 haplotype of this strain.

Influence of splenectomy upon immunologic reactivity of patients with Hodgkin's disease.

Since most patients with Hodgkin's disease benefit from splenectomy, a study was designed to explore whether these beneficial effects could be attributed to the recovery of patients' immunologic reactivity. Using a series of ordinary skin test (PPD-tuberculine, Varidase and Candidin) determination of absolute T and B lymphocyte counts in peripheral blood and their mitogenic responsiveness, assessment of immunologic reactivity was performed in 28 Hodgkin's disease patients, prior to and 14 days after splenectomy. The results showed that overall immunologic reactivity of these patients was suppressed as judged by low absolute lymphocyte counts (1747.2 +/- 171.9), lower counts of T (592.0 +/- 92.1) and B cells (295.9 +/- 40.5) and their poor capacity to respond to phytohemagglutinin (PHA) (20342.6 +/- 3662.8 cpm), although the reactivity towards skin test antigens seemed to be well preserved. After splenectomy the reactivity improved, absolute lymphocyte counts raised to 2654.9 +/- 468.8 and were parallelled by an increase in T (936.7 +/- 138.0) and B cell counts (402.2 +/- 81.2). PHA reactivity recovered as well (26965.5 +/- 4035.6 cpm), however, its remained lower than in control cultures. Furthermore, the immunocompetence of patients' spleens was assessed. The possible influence of some suppressive mechanisms such as serum-blocking factor and prostaglandins is discussed.

BCG immunotherapy in previously treated malignant melanoma patients.

Eighteen patients with histologically confirmed metastatic malignant melanoma were treated with intradermal BCG. Before starting immunotherapy their immunocompetence was tested in vivo and in vitro. Four of 15 (27%) achieved complete regression, and two (13%) a regression of more than 50%, after systemic BCG. All three patients treated with perinodular injection of BCG had complete regression of treated as well as some of untreated nodules. Treatment was unsuccessful in 9 patients. Five of them (33%) had a disease stabilisation for more than 5 months. The results of BCG immunotherapy were compared with those of chemotherapy alone. The immunotherapy patients had longer remissions and survived longer than those treated by chemotherapy. All patients had repeat skin tests with PPD after BCG but showed no significant improvement in tuberculin reactivity. Despite, their clinical condition often being improved after BCG. We conclude that BCG may be of considerable benefit to malignant melanoma patients.

A preliminary report of a pilot trial in adjuvant chemotherapy of primary melanoma.

It is well known that level of skin invasion and tumor thickness are significant prognostic factors in the evolution of primary melanoma. The prognosis of primary melanoma Clark III to V skin invasion level and more than 1.5 mm thick confirms this statement. Even the prophylactic dissection of regional lymph nodes has not improved results. In an attempt to obtain better results in the treatment of primary melanomas, a pilot trial was carried out combining surgery and adjuvant chemotherapy. A group of 21 patients with Clark III, IV and V level primary melanoma who underwent adjuvant polychemotherapy (velba + dactinomycin + procarbazine) for 1 year after surgery showed a very low incidence of recurrences (5%) after 24 months of observation. The historical control group, with the same level of tumor skin invasion, treated only surgically had in the same follow-up period a recurrence rate of 65%. This difference was statistically significant (p less than 0.01). All patients who received adjuvant chemotherapy survived 2 years whereas survival was 77% (p less than 0.05) in the surgical historical control group. Favorable results with the same protocol of adjuvant chemotherapy were not obtained in the group of 16 patients with stage II melanoma when compared with primary tumors. However, 4 recurrences were observed after 12 months of observation; toxic side effects of adjuvant chemotherapy were mild and tolerable. Considering the insufficient number of clinical trials with adjuvant chemotherapy, as well as sometimes controversial results, further randomized clinical studies are needed to establish the actual value of this conbined method in the treatment of primary melanoma with a high risk of dissemination.

In vitro detection of cellular immunity to melanoma antigens in man by the monocyte spreading inhibition test.

In vitro inhibition of monocyte spreading (a correlate of cellular immunity) was used to detect cell-mediated immune reactions of melanoma patients to specific melanoma antigens. Two soluble preparations of human melanoma antigens (MA-1 and MA-2) and one of a breast carcinoma (BCA) were prepared. The preparations were incubated in vitro with mononuclear cells isolated from the blood of 24 patients with melanoma, six patients with malignancies other than melanoma and 14 healthy donors. Spreading of monocytes from healthy donors was not inhibited by either MA-1 and MA-2 or BCA. MA-1 and MA-2 significantly inhibited the spreading of monocytes from patients with melanoma, while monocytes from patients with other malignancies were not affected. Spreading of monocytes from patients with melanoma was inhibited by the preparation of BCA. We conclude that inhibition of monocyte spreading can detect, in vitro, a cellular immune reaction to specific melanoma antigens in patients with melanoma.