Differences in the action and metabolism between retinol and retinoic acid in B lymphocytes.

We have previously reported on the dependency of activated B lymphocytes for retinol. Here we confirm and extend these findings that cells deprived of retinol perish in cell culture within days, displaying neither signs of apoptosis nor of cell cycle arrest. Cell death can be prevented by physiological concentrations of retinol and retinal, but not by retinoic acid or three synthetic retinoic acid analogues. To exclude the possibility that retinoic acid is so rapidly degraded as to escape detection, we have tested its stability in intra- and extracellular compartments. Contrary to expectation, we find that retinoic acid persists for longer (t 1/2 3 d) in cultures than retinol (t 1/2 1 d). Furthermore, despite the use of sensitive trace-labeling techniques, we cannot detect retinoic acid or 3,4-didehydroretinoic acid among retinol metabolites. However, retinol is converted into several new retinoids, one of which has the ability to sustain B cell growth in the absence of an external source of retinol, supporting the notion of a second retinol pathway. We have also determined which of the known retinoid-binding proteins are expressed in B lymphoblastoid cells. According to results obtained with polymerase chain reaction-assisted mRNA detection, they transcribe the genes for cellular retinol- and cellular retinoic acid-binding proteins, for the nuclear retinoic acid receptors, RAR-alpha, -gamma, and RXR-alpha, but not RAR-beta. Our findings that B cells do not synthesize retinoic acid or respond to exogenous retinoic acid on the one hand, but on the other hand convert retinol to a novel bioactive form of retinol, suggest the existence of a second retinoid pathway, distinct from that of retinoic acids.

Effect of swainsonine on stimulation and cell cycle progression of human lymphocytes.

The indolizidine alkaloid swainsonine (SW) is an inhibitor of lysosomal alpha-mannosidase reported to have antimetastatic activity in animal models. The cells grown in its presence develop truncated (hybrid) surface oligosaccharides that may alter their functional properties dependent on interactions of various ligands with membrane receptors. In the present study we observe that SW enhances stimulation of human lymphocytes induced by suboptimal concentration of concanavalin A. The enhancement is manifested by an increased proportion of cells undergoing transition from G0 (G1Q) to G1 and progressing through the cell cycle (S + G2 + M). In contrast, SW suppresses stimulation of lymphocytes by phytohemagglutinin, and the degree of suppression is greater when measured by the number of cells progressing through the cell cycle (S + G2 + M) than by the proportion of cells entering G1 phase. The suppression remains evident even when SW is added 12 h after phytohemagglutinin, suggesting that SW modifies membrane receptors that develop in G1 and are necessary for cell entrance to S phase. The modification of receptors by SW thus up-regulates stimulation by concanavalin A and down-regulates stimulation by phytohemagglutinin. SW has no effect on lymphocyte stimulation induced by OKT3 monoclonal antibody or on the progression of cells from three leukemic cell lines, HL-60, L1210, and MOLT-4, through the cell cycle. The present data are compatible with the possibility that the reported suppression of the growth of metastatic mouse tumors by SW may be due to the immunomodulatory properties of this alkaloid.

Immunohistochemical characterization of two monoclonal antibodies, P25.48 and P25.91, which define a new prostate-specific antigen.

We have generated two new mouse monoclonal antibodies against prostate cancer. P25.48 (IgG3) and P25.91 (IgG2a) were derived from a fusion using fresh prostate cancer cells as the immunogen. Initial screening was performed by indirect immunofluorescence on frozen tissue sections of prostate cancer specimens. The specificity analysis was performed by indirect immunoperoxidase on frozen sections of normal tissues and benign and malignant prostate tissues. P25.48 and P25.91 did not react with any benign prostatic tissues (0 of 17), but reacted with a subset of the malignant prostatic tissues. Five specimens of well-differentiated carcinoma were tested and did not react with P25.48 or with P25.91. Of 16 higher grade specimens, nine reacted with both P25.48 and P25.91, one reacted with P25.48 only, and one reacted with P25.91 only. In most positive cases, the reactivity was heterogenous. P25.48 and P25.91 showed a very restricted pattern of reactivity in nonprostatic tissues. Of 50 normal specimens, only one breast specimen showed some reactivity. None of the nine fetal tissues or of the 15 malignant tissues tested reacted with these monoclonal antibodies. The pattern of reactivity of P25.48 and P25.91 suggests that they recognize the same antigen. This antigen is selectively expressed by malignant prostatic epithelium. In addition, it appears to be distinct from all other previously described prostate cancer-associated antigens.

Characterization of FAP-1 expression and function in thyroid follicular cells.

Human thyrocytes are resistant to Fas-mediated programmed cell death (PCD). It has been reported that a labile protein inhibitor is involved in the protection of thyrocytes from PCD, and its action can be reversed by incubation of thyrocytes with cycloheximide (CHX) during treatment with agonist anti-Fas Ab. Fas-associated phosphatase-1 (FAP-1) is a protein that has been shown to interact with the negative regulatory domain of Fas and block Fas-mediated apoptosis in FAP-1 transfected Jurkat cells. We investigated the possibility that FAP-1 might be involved in protection against Fas-mediated PCD in human thyrocytes. FAP-1 mRNA was detected in primary thyrocytes using a ribonuclease protection assay. The presence of FAP-1 protein was confirmed by immunohistochemical staining and flow cytometry using a polyclonal anti-FAP-1 Ab. FAP-1 protein also disappeared from thyroid cells in response to CHX. To determine whether FAP-1 is a functional inhibitor of PCD in thyrocytes, we incubated thyrocytes with synthetic SLV (Ac-SLV) tripeptide to compete with Fas for interaction with FAP-1. Thyrocytes treated with Ac-SLV tripeptide showed significantly increased cell death as compared to cells treated with control tripeptide. In addition, in the presence of a suboptimal concentration of CHX, the Ac-SLV tripeptide yielded a strong, synergistic increase in Fas-mediated PCD as compared to thyrocytes treated with control tripeptide. These results implicate FAP-1 as a regulator of Fas-induced PCD in thyrocytes.

1Alpha,25-dihydroxyvitamin D3 up-regulates Bcl-2 expression and protects normal human thyrocytes from programmed cell death.

Apoptosis is thought to play an important role in the pathogenesis of autoimmune thyroid disease. 1alpha,25-dihydroxyvitamin D3 (VD3) has been shown to suppress several autoimmune diseases. However, the mechanism by which VD3 has these effects is not known. We evaluated the alterations in apoptosis, induced by VD3. Thyrocytes were treated with VD3, and the expression of the Bcl-2 family molecules was studied at both the messenger RNA and protein levels. It was found that VD3 significantly induced the expression of Bcl-2 messenger RNA and protein in thyrocytes but had no effect on the expression of Bcl-xl and Bax. The increase in Bcl-2 expression, mediated by VD3, correlated with protection of thyrocytes against the induction of apoptosis by either staurosporine or UV irradiation. VD3-induced increases in the expression of Bcl-2 could be mimicked by VD3 analogs with high nuclear receptor affinity, but not by analogs only with nongenomic actions. These data indicate a role for Bcl-2 in the regulation of apoptosis in thyrocytes and raise the possibility that VD3 or its agonists may have therapeutic benefit in thyroid disorders.

Fas (CD95) expression is up-regulated on papillary thyroid carcinoma.

Thyrocyte apoptosis signaled through the Fas receptor has been proposed as a mechanism for the cytotoxicity observed in thyroiditis, but the role the Fas pathway plays in thyroid cancer is not known. We examined Fas expression in thyroid tissue derived from patients with papillary carcinoma and follicular cancer. More intense immunohistological staining for the Fas protein was observed on papillary cancer cells as compared with adjacent normal follicles. To further characterize the expression of Fas in papillary cancer, paired normal and cancerous thyroid tissues were obtained at thyroidectomy from several donors, digested, and placed into cell culture. Messenger RNA was analyzed by ribonuclease protection assays, and protein was identified by flow cytometry. Fas expression was detected at levels up to 3-fold higher in cancerous thyrocytes compared with paired normal cells. To determine whether the expressed Fas antigen was functional, thyrocytes were treated with a monoclonal IgM anti-Fas antibody (clone CH11; Upstate Biotechnology, Inc., Lake Placid, NY) in the presence of interferon-gamma and cycloheximide. Whereas both normal and cancerous thyrocytes were induced to die after this treatment, the cancerous thyrocytes were more sensitive to anti-Fas antibody. This work demonstrates that the Fas antigen is expressed and functional on papillary thyroid cancer cells and this may have potential therapeutic significance.

Studies on binding of HIV-1 p24gag peptide to HLA-Cw3+ cells.

Human major histocompatibility complex class I antigens, HLA-C, are expressed on the cell surface at approximately a tenfold lower level than HLA-A and -B. We hypothesized that the expression of HLA-C is limited by the quantity of high affinity peptides which bind to these molecules, thus allowing only a small fraction of HLA-C molecules to be transported and/or to remain stable on the cell surface. If this assumption is correct, then the addition of exogenous peptide should increase cell surface HLA-C expression. To verify the hypothesis, we pulsed lymphoblastoid cell line PAJ (HLA-Cw3+) with synthetic HIV-1 p24gag 145-152 peptide, known to be presented to T-lymphocytes by HLA-Cw3 molecule. PAJ (HLA-Cw3+) cells bound approximately two times more of the peptide than HAJ (HLA-Cw3-), and four times more than 500/C9 (HLA-Cw3-) cells. Accordingly, overnight pulsing of PAJ cells with the p24gag 145-152 peptide caused an increase in class I HLA expression detected on the cell surface by flow cytofluorimetric analysis with anti-HLA-B,C monoclonal antibodies but not by anti-HLA-A antibody. In contrast, HLA-Cw3- cells treated in the same manner did not show any increase of HLA class I expression. Our data suggest that low concentration of high affinity peptides within the cell may be one of the factors limiting cell surface expression of HLA-C molecules.

2-Methoxyestradiol, an endogenous estrogen metabolite, induces thyroid cell apoptosis.

The etiology of autoimmune thyroid diseases is unclear; however, the extreme female predominance suggests that sex hormones may have a pathogenic role. 2-Methoxyestradiol (2-ME) is present in the serum of women during the ovulatory and luteal phases of the menstrual cycle, and during pregnancy. We investigated the actions of 2-ME and estrogen on thyroid follicular cells. 2-ME induced dramatic changes in cell morphology and decreased the viability of the cells, as well as disrupted the structural integrity of cultured thyroid follicles. Flow cytometric analysis showed that 2-ME halted cell proliferation by arresting the cells in the G2/M cell-cycle compartment. Prolonged exposure to 2-ME led to apoptosis and to increased release of the autoantigen thyroid peroxidase (TPO). 17beta-estradiol failed to produce a similar effect even in 40-fold molar excess to 2-ME. Co-treatment with estrogen receptor antagonists did not alter the 2-ME effect, indicating that 2-ME was not operating through a classic nuclear estrogen receptor. In conclusion, this study indicates that 2-ME induces G2/M cycle arrest, apoptosis and the disruption of thyroid follicles. This process results in the release of thyroid antigens that may play a role in high incidence of thyroid autoantibodies and autoimmune thyroid disease in women.

In vitro bromodeoxyuridine labeling of malignant neoplasms. A comparative study with flow cytometry cell-cycle analysis.

Proliferative fraction has been defined as an independent prognostic marker for some malignant neoplasms. An estimate of the proliferative fraction can be obtained by cell-cycle analysis of flow cytometric DNA measurements. However, overlapping DNA distributions of aneuploid neoplasms are difficult to analyze, even with sophisticated computer programs, and results are not always reproducible. Bromodeoxyuridine (BrdUrd) labeling followed by immunohistochemical detection has been proposed as an alternative, simple, and accurate method for identifying and counting DNA synthesizing cells. In vitro BrdUrd labeling was performed on 87 tumors, including 35 lung cancers, 25 breast carcinomas, and 27 tumors of other origin. Results were compared with flow cytometric S-phase estimates in 46 cases. Mean BrdUrd labeling of lung tumors was 8.5% +/- 5.2%, compared with a mean flow cytometric S-phase fraction of 20% +/- 18.4%. Mean BrdUrd labeling of breast carcinomas was 6.8% +/- 3.8%, compared with a mean flow cytometric S-phase fraction of 12.5% +/- 9.9%. In both tumor types, BrdUrd labeling correlated well with histologic grade. Correlation between BrdUrd labeling and flow cytometric S-phase estimates were generally poor, particularly with aneuploid tumors.

Optimization of in situ cellular ELISA performed on influenza A virus-infected monolayers for screening of antiviral agents.

Viral susceptibility testing has been traditionally performed by the plaque reduction assay (PRA) which is laborious, time consuming, relatively expensive, and requires subjective input by the reader. An in situ cellular enzyme-linked immunosorbent assay (ELISA) has been developed with the potential to overcome many of the limitations of PRA and has been applied to a variety of viruses. This study establishes the specific conditions necessary for susceptibility testing of influenza A virus to antiviral agents such as amount of inoculum size, duration of incubation, fixative type, and cell number; factors which are critical to the performance of the in situ cellular ELISA. In situ cellular ELISA was found to correlate strongly with the plaque assay (PA) (R2 = 0.997, P < 0.002). Both assays were applied to test the susceptibility of influenza A virus to a new antiviral emulsion agent and yielded comparable data. The optimized in situ cellular ELISA can serve as a reliable assay for the rapid screening of large numbers of antiviral agents.

Prevention of murine influenza A virus pneumonitis by surfactant nano-emulsions.

Non-ionic surfactant nano-emulsions have extensive anti-microbial activity and are biocompatible with skin and mucous membranes at effective concentrations. Two nano-emulsion formulations (8N8 and 20N10) made from soybean oil, tributyl phosphate and Triton X-100, were tested for their ability to prevent murine influenza virus pneumonia in vivo. In the initial study, CD-1 mice were administered various dilutions of the nano-emulsions intranasally, and safe dosages and concentrations were determined. Non-toxic concentrations of the nano-emulsions were then mixed with influenza virus and applied to the nares of mice. Animals receiving mixtures of two different emulsions (8N8 or 20N10) and a LD50 of virus survived the challenge without evidence of viral infection. To determine if the nano-emulsions could prevent influenza virus infection in vivo when used as a prophylactic treatment, the nano-emulsions (8N8 at 1.0% and 20N10 at 1.0% or 0.2%) were applied to mouse nares 90 min before exposure to 5x10(5) p.f.u./ml virus by nebulized aerosol. Animals pretreated with the nano-emulsions had significantly decreased clinical signs of infection. Only 26.0% (8N8 at 1.0%), 31.25% (20N10 at 1.0%) and 37.0% (20N10 at 0.2%) of animals pretreated with nano-emulsion died from pneumonitis, whereas >80.0% of mock pretreated animals succumbed to infection (P<0.005). These findings suggest that non-ionic surfactant nano-emulsions have therapeutic potential for the prevention of influenza virus infection in vivo.

Age-related changes in PHA and allogeneic reactivity of human T lymphocytes.

In fifty-four healthy individuals aged from 22 to 53 the response of lymphocytes to PHA stimulation and to stimulation with allogeneic lymphocytes were determined. A linear age-related decrease in reactivity to PHA (r = 0.500, P less than 0.01), as well as to allogeneic stimulation (r = 0.351, P less than 0.01) was found. The correlation between these two parameters of T lymphocyte function was weak (r = 0.207, not significant).

DR antigens as a guide in the search for Dw homozygous typing cells (HTC).

Advantage has been taken from association between DR and Dw genes in the search for homozygous typing cells (HTC). From population data it was calculated that the probability of Dw homozygosity should be about 10 times higher among persons with one identified DR antigen than in a random population. To prove this assumption cells from 11 persons with one DR antigen were tested in different MLR combinations and 5 from them gave reaction patterns typical for HTC. These cells revealed ability to discriminate between high and low MLR responses as indicated by the bimodal or asymmetric curves of distribution of the results.

MLR-stimulating properties of five homozygous typing cells found in an outbred population.

In the characterization of stimulating properties of five HTC's two features were taken into consideration, the reproducibility of stimulation results and the individual cut-off level at which reactions positive for Dw antigen are separated from negative reactions. The reproducibility was characterized by the number of repetitions necessary to diminish the standard error to 10 DNV. Depending on HTC 2-8 repetitions were found to be necessary. The individual cut-off levels were determined with the help of maximal correlation between Dw and DR antigen typing. For different HTC's the levels varied from 22 to 57 DNV.

The MLC response of patients with infectious mononucleosis to Epstein-Barr virus-transformed cells.

Lymphocytes of 6 patients with infectious mononucleosis were found to be capable of responding in mixed lymphocyte culture (MLC) to Epstein-Barr virus (EBV)--transformed lymphoblastoid cell lines and Raji cells, whereas their response to PWM-induced blasts and non-T cells was significantly depressed. This means that despite the suppression of cellular reactivity, observed in infectious mononucleosis, the specific response to EBV-infected cells remains unaffected.

The Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) as stimulating cells in mixed lymphocyte culture (MLC).

The MLC response of normal lymphocytes to different kinds of EBV-transformed LCL was investigated. The response to allogeneic LCL cells remarkably exceeded the response to xenogeneic and autologous LCL cells. Stimulation performed with EBV-transformed and normal cells obtained from the same donors showed a correlation in the reaction levels (Q = 0.434). This incomplete correlation was not improved by the reduction of the number of stimulating LCL cells. In MLC stimulated with LCL no statistically significant differences between responses to 2,1 or alloantigens were found.

Blood transfusions and the development of cytotoxic antibodies in dialyzed patients.

In our work we investigated factors which can influence the alloimmunization and especially the ranges of cytotoxic antibodies that develop in patients on dialysis awaiting their first graft. Up to 15 liters of administered blood the proportion of immunized patients is rather stabile, not exceeding 50%. After further transfusions the percentage of immunized patients is growing rapidly reaching about 85%. Hyperimmunization, marked by cytotoxic antibodies with more than 80% panel reactivity is significantly more frequent in patients who obtained 15 and more liters of blood. In females the hyperimmunization is about 4 times more frequent than in males.

A novel surfactant nanoemulsion with a unique non-irritant topical antimicrobial activity against bacteria, enveloped viruses and fungi.

A novel non-ionic surfactant nanoemulsion designated 8N8 has been tested for its biocidal activity. One percent 8N8 produced effective bactericidal activity against Bacillus cereus, Bacillus subtilis, Haemophilus influenzae, Neisseria gonorrhoeae, Streptococcus pneumoniae, and Vibrio cholerae in 15 minutes. In contrast, most enteric gram-negative bacteria were resistant to 8N8. One percent 8N8 was also virucidal within 15 minutes for all tested enveloped viruses, including Herpes simplex type 1, influenza A and vaccinia viruses. One percent 8N8 also demonstrated fungistatic activity on Candida albicans. The rapid and non-specific inactivation of vegetative bacteria and enveloped viruses, in addition to its fungistatic activity and low toxicity in experimental animals, makes 8N8 a potential candidate for use as a topical biocidal agent.

Safety and immunogenicity of a novel nanoemulsion mucosal adjuvant W805EC combined with approved seasonal influenza antigens.

BACKGROUND: Improving the systemic and mucosal immune response following intranasal vaccination could enhance disease protection against respiratory pathogens. We assessed the safety and immunogenicity of a novel nanoemulsion mucosal adjuvant W(80)5EC combined with approved seasonal influenza antigens. METHODS: This was a first-in-human Phase I study in 199 healthy adult volunteers randomized to receive a single intranasal administration of 5%, 10%, 15% or 20% W(80)5EC, combined with 4 or 10 µg strain-specific Fluzone(®) HA, compared with intranasal PBS, intranasal Fluzone(®), or 15 ug strain-specific intramuscular Fluzone(®). Safety was evaluated by physical examination, laboratory parameters, symptom diaries, and adverse event reports. Serum HAI titers and nasal wash IgA were assessed at baseline as well as 28 and 60 days after vaccination. RESULTS: W(80)5EC adjuvant combined with seasonal influenza antigens was well tolerated without safety concerns or significant adverse events. The highest dose of 20% W(80)5EC combined with 10 µg strain-specific HA elicited clinically meaningful systemic immunity based on increases in serum HAI GMT and ≥ 70% seroprotection for all 3 influenza strains, as well as a rise in antigen-specific IgA in nasal wash specimens. CONCLUSIONS: W(80)5EC adjuvant was safe and well tolerated in healthy adult volunteers and elicited both systemic and mucosal immunity following a single intranasal vaccination.