Association between Sézary T cell-activating factor, Chlamydia pneumoniae, and cutaneous T cell lymphoma.

Sézary T cell-activating factor (SAF) was originally defined as an inducer of functional interleukin-2 (IL-2) receptors on normal and malignant T cells in patients suffering from Sézary syndrome. In fact, a combination of SAF and IL-2 stimulated the propagation of T cell lines from the peripheral blood mononuclear cells (PBMC) of those patients, with approximately one third of those cell lines containing the predominant malignant clone as determined via cytogenetic and/or T cell receptor gene rearrangement analysis. Although the primary source of SAF was mitogen-stimulated PBMC of a patient with Sézary syndrome, we were unable to isolate the gene encoding SAF from eukaryotic libraries. However, we observed SAF activity in the cytoplasm of one of the malignant cell lines in a complex containing RNA and DNA. This observation led us to consider the possibility that SAF is not of eukaryotic origin. Intracellular pathogens replicate in the cytoplasm of host cells and contain proteins, DNA, and RNA. Using a panel of antichlamydial antibodies with confirmation from polymerase chain reaction primers, we found that most patients with mycosis fungoides were positive for these determinants. Immunoelectron microscopy and protein blotting further confirmed antibody reactivity. We showed that Chlamydia pneumoniae were capable of infecting normal human keratinocytes in culture. We also demonstrated that C. pneumoniae antigen expression was associated with active disease because these determinants were not expressed after psoralen and ultraviolet A therapy. We hypothesize that chronic infection by C. pneumoniae leads to expansion of C. pneumoniae-specific T cells, thereby potentiating the development of cutaneous T cell lymphoma.

Sézary T-cell activating factor is a Chlamydia pneumoniae-associated protein.

We previously identified a protein that was stimulatory for malignant Sézary T cells, termed Sézary T-cell activating factor (SAF). However, the identity of this protein has not been fully elucidated, nor has it's role been determined in the pathogenesis of cutaneous T-cell lymphoma (CTCL). The basis for epidermotropism and proliferation of malignant cells in the skin of patients with CTCL is unknown. Using a monoclonal antibody inhibitory for SAF activity, we demonstrated that SAF is present in the skin of 16 of 27 samples from patients with mycosis fungoides, the predominant form of CTCL. In this report, the SAF determinant is demonstrated to be associated with Chlamydia pneumoniae bacteria by immunohistochemistry, immunoelectron microscopy, and culture analysis. Reactivity of antibodies against an outer membrane protein of C. pneumoniae or against the lipopolysaccharide of Chlamydiae spp. demonstrated that these determinants are coexpressed in 90% of the SAF-positive samples. We confirmed the presence of C. pneumoniae DNA and RNA in the skin by PCR and reverse transcription-PCR and by sequence analysis of the PCR products. The expression of the C. pneumoniae antigens and SAF appears to be associated with active disease in that C. pneumoniae antigens were absent or greatly diminished in the skin of three patients examined after Psoralen and long-wave UVA radiation treatment. Our results suggest that SAF is a Chlamydia-associated protein and that further investigation is warranted to determine whether SAF and C. pneumoniae play a role in the pathogenesis of CTCL.

Increased immunogenicity of tumor vaccines complexed with anti-Gal: studies in knockout mice for alpha1,3galactosyltransferase.

A major prerequisite for the success of tumor vaccines is their effective uptake by antigen-presenting cells (APCs) and transport of these APCs to the draining lymph nodes, where the processed and presented tumor-associated antigens activate tumor-specific naive T cells. We previously suggested that the immunogenicity of autologus tumor vaccines in humans may be augmented by engineering vaccinating tumor cell membranes to express alpha-galactosyl (alpha-gal) epitopes (i.e., Galalpha1,3Galbeta1,4GlcNAc-R). Subsequent in situ binding of natural anti-Gal IgG molecules to these epitopes would result in the formation of immune complexes that target tumor vaccines for uptake by APCs, via the interaction of the Fc portion of anti-Gal with Fcgamma receptors on APCs. This hypothesis was tested in a unique experimental animal model of knockout mice for alpha1,3galactosyltransferase (alpha1,3GT) and the mouse melanoma B16-BL6 (referred to here as BL6). Like humans, these mice lack alpha-gal epitopes and produce anti-GaL BL6 melanoma cels are highly tumorigenic, and like human tumor cells, they lack alpha-gal epitopes. Expression of alpha-gal epitopes on these melanoma cells was achieved by stable transfection with alpha,3GT cDNA. The transfected melanoma cells (termed BL6alphaGT) express approximately 2 x 10(6) alpha-gal epitopes per cell and readily form immune complexes with anti-Gal. Vaccination of the mice with 2 x 10(6) irradiated melanoma cells that express alpha-gal epitopes, followed by challenge with 0.5 x 10(6) live parental melanoma cells, resulted in protection for at least 2 months (i.e, no tumor growth) in one-third of the mice, whereas all mice immunized with irradiated parental melanoma cells developed tumors 21-26 days post-challenge. The proportion of protected mice doubled when the mice were immunized twice with irradiated melanoma cells expressing alpha-gal epitopes and challenged with 0.2 x 10(6) live BL6 cells. Histological studies on the developing tumors in challenged mice that were immunized with melanoma cells expressing alpha-gal epitopes demonstrated extensive infiltration of T lymphocytes and macrophages, whereas no mononuclear cell infiltrates were observed in tumors of mice immunized with parental tumor cells. Overall, these studies imply that immunization of alpha1,3GT knockout mice with BL6 melanoma cells that express alpha-gal epitopes elicits, in a proportion of the population, protective immune response against the same tumor lacking such epitopes. These studies further suggest that similar immunization of cancer patients with autologous tumor vaccines that are engineered to express alpha-gal epitopes may increase the immune response to autologous tumor-associated antigens and, thus, may elicit immune-mediated destruction of metastatic cells expressing these antigens.

Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain.

We assessed whether the intracellular bacterium Chlamydia pneumoniae was present in post-mortem brain samples from patients with and without late-onset Alzheimer's disease (AD), since some indirect evidence seems to suggest that infection with the organism might be associated with the disease. Nucleic acids prepared from those samples were screened by polymerase chain reaction (PCR) assay for DNA sequences from the bacterium, and such analyses showed that brain areas with typical AD-related neuropathology were positive for the organism in 17/19 AD patients. Similar analyses of identical brain areas of 18/19 control patients were PCR-negative. Electron- and immunoelectron-microscopic studies of tissues from affected AD brain regions identified chlamydial elementary and reticulate bodies, but similar examinations of non-AD brains were negative for the bacterium. Culture studies of a subset of affected AD brain tissues for C. pneumoniae were strongly positive, while identically performed analyses of non-AD brain tissues were negative. Reverse transcription (RT)-PCR assays using RNA from affected areas of AD brains confirmed that transcripts from two important C. pneumoniae genes were present in those samples but not in controls. Immunohistochemical examination of AD brains, but not those of controls, identified C. pneumoniae within pericytes, microglia, and astroglia. Further immunolabelling studies confirmed the organisms' intracellular presence primarily in areas of neuropathology in the AD brain. Thus, C. pneumoniae is present, viable, and transcriptionally active in areas of neuropathology in the AD brain, possibly suggesting that infection with the organism is a risk factor for late-onset AD.

The prognostic significance of delayed hypersensitivity to dinitrochlorobenzene and mechlorethamine hydrochloride in cutaneous T cell lymphoma.

Recent studies suggest that cells elaborating type 1 cytokines are important mediators of anti-tumor cell-mediated immunity in cutaneous T cell lymphoma. Type 1 cell-mediated immune responsiveness was assessed in 276 patients with cutaneous T cell lymphoma (mycosis fungoides and Sézary syndrome) using 2,4-dinitrochlorobenzene (DNCB) skin testing as part of the initial evaluation. The overall rate of sensitization after one and two DNCB challenges was 32% and 67%, respectively, which is much decreased compared with the expected rate of more than 95% for normal individuals. Moreover, the frequency of DNCB sensitization and allergic contact dermatitis to topically applied mechlorethamine decreased with advancing stage of disease. In addition to the expected strong correlation with stage, we observed that patients who were DNCB test positive were significantly less likely to experience disease progression and had a better overall prognosis compared with DNCB-negative patients. These results support the concept that cell-mediated responses are important in cutaneous T cell lymphoma, and that augmentation of these responses would be therapeutically beneficial.

Use of serum soluble interleukin-2 receptor levels to monitor the progression of cutaneous T-cell lymphoma.

BACKGROUND: The serum concentration of soluble alpha chain of the interleukin-2 receptor (sIL-2R) correlates with tumor burden in cutaneous T-cell lymphoma (CTCL). Therefore the sIL-2R level may be useful to monitor the condition of patients treated with extracorporeal photopheresis or other treatments. OBJECTIVE: Our goal was to determine the utility of serum sIL-2R as a test in monitoring of patients with advanced CTCL. METHODS: Serum sIL-2R was measured serially in 36 patients with advanced CTCL treated with extracorporeal photopheresis and other modalities (interferon alfa, methotrexate, topical nitrogen mustard, electron beam). RESULTS: Serum concentrations of sIL-2R as well as lactate dehydrogenase (LDH) correlated strongly with lymph node size, but only sIL-2R correlated significantly with the severity of skin manifestations in erythrodermic patients. In addition, serum sIL-2R, but not LDH, was significantly higher in patients with nodal involvement. The level of sIL-2R also was significantly higher in patients with large-cell transformation in the skin or lymph nodes compared with patients without transformed disease. During treatment, serum concentrations of both serum sIL-2R and LDH correlated with changes in clinical status, but only sIL-2R showed statistically significant differences in mean levels for different relative global response scores. Pretreatment levels of both sIL-2R and LDH correlated significantly with survival, but only sIL-2R retained significance when both were entered into the Cox proportionate hazards model. CONCLUSION: The concentration of serum sIL-2R correlates well with disease status and is more useful than LDH or Sézary cell counts to monitor clinical change in patients with advanced CTCL. Moreover, our data suggest that sIL-2R is produced at a relatively low rate by tissue-based lymphoma cells, and that large-cell transformation in CTCL results in marked increase in sIL-2R production in some patients.

Gastrin induces IP3 formation through phospholipase C gamma 1 and pp60c-src kinase.

We have previously reported that gastrin induces a rapid and transient tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) in association with inositol 1,4,5-trisphosphate (IP3) formation in rat colonic epithelial cells (34). In this study, we demonstrate that gastrin regulates IP3 formation mainly through PLC gamma 1 isozyme. Immunoblotting analysis revealed the expression of PLC beta 3 and -gamma 1, but not PLC beta 1, -beta 2, or -beta 4 in the rat colonic epitheliums. To explore what PLC isozyme(s) modulates gastrin effect on IP3, immunoneutralizing antibody to PLC beta 1, -beta 3, or -gamma 1 was introduced into the colonic cells using a lipid carrier. The gastrin-stimulated increase in IP3 concentration was specifically prevented by anti-PLC gamma 1 but not by anti-PLC beta 1 or -beta 3 antibody. Immunoprecipitation assays have also revealed that gastrin promoted an increase in tyrosine phosphorylation and co-precipitation of a 60 kDa src kinase with PLC gamma 1. Administration of antibody specific to pp60c-src into the colonic cells prevented the gastrin-stimulated increases in IP3. Tyrosine phosphorylation of PLC gamma 1 may be a major mechanism through which gastrin regulates IP3 level in the colonic cells. Pretreatment of cells with the tyrosine kinase inhibitor genistein abrogated gastrin's effect on IP3, while extended pretreatment with pertussis toxin, a G-protein inhibitor, did not affect the ability of gastrin to stimulate IP3 formation. Colonic cells expressed the G alpha i subunits1-3; however, immunoblotting analysis did not reveal any difference in G alpha i proteins' expression between control and gastrin treated cells. The results provide direct evidence that gastrin regulates IP3 level by a signaling mechanism that involves PLC gamma 1 and pp60c-src kinase.

Difficult or impossible ventilation after sufentanil-induced anesthesia is caused primarily by vocal cord closure.

INTRODUCTION: Opioid-induced rigidity often makes bag-mask ventilation difficult or impossible during induction of anesthesia. Difficult ventilation may result from chest wall rigidity, upper airway closure, or both. This study further defines the contribution of vocal cord closure to this phenomenon. METHODS: With institutional review board approval, 30 patients undergoing elective cardiac surgery participated in the study. Morphine (0.1 mg/kg) and scopolamine (6 microg/kg) given intramuscularly provided sedation along with intravenous midazolam as needed. Lidocaine 10% spray provided topical anesthesia of the oropharynx. A fiberoptic bronchoscope positioned in the airway photographed the glottis before induction of anesthesia A second photograph was obtained after induction with 3 microg/kg sufentanil administered during a period of 2 min. A mechanical ventilator provided 10 ml/kg breaths at 10/min via mask and oral airway with jaw thrust. A side-stream spirometer captured objective pulmonary compliance data. Subjective airway compliance was scored. Pancuronium (0.1 mg/kg) provided muscle relaxation. One minute after the muscle relaxant was given, a third photograph was taken and compliance measurements and scores were repeated. Photographs were scored in a random, blinded manner by one investigator. Wilcoxon signed rank tests compared groups, with Bonferroni correction. Differences were considered significant at P < 0.05. RESULTS: Twenty-eight of 30 patients exhibited decreased pulmonary compliance and closed vocal cords after opioid induction. Two patients with neither objective nor subjective changes in pulmonary compliance had open vocal cords after opioid administration. Both subjective and objective compliances increased from severely compromised values after narcotic-induced anesthesia to normal values (P = 0.000002) after patients received a relaxant. Photo scores document open cords before induction, progressing to closed cords after the opioid (P = 0.00002), and opening again after a relaxant was administered (P = 0.00005). CONCLUSION: Closure of vocal cords is the major cause of difficult ventilation after opioid-induced anesthesia.

Porcine and bovine cartilage transplants in cynomolgus monkey: I. A model for chronic xenograft rejection.

Transplantation of discordant xenograft tissues usually results in antibody-mediated hyperacute rejection response. It has been speculated that because cartilage has a limited vascular, neural, and lymphatic supply, it might be immunologically privileged and may not undergo hyperacute or chronic rejection. Moreover, porcine and bovine cartilage were found to express very low amounts of alpha-galactosyl epitopes (Gal alpha1-3Gal beta1-4GlcNAc-R). To evaluate animal cartilage for possible human transplantation, xenograft meniscal cartilage was transplanted from pigs and cows into the suprapatellar pouches of six cynomolgus monkeys (group 1). In a second group of six monkeys (group 2), porcine meniscal cartilage and porcine articular cartilage plugs were evaluated. During the 2-month evaluation period in group 1, all monkeys displayed an extensive humoral response to the xenograft, as indicated by the increase in production of antibodies against bovine and porcine cartilage. Upon explant, all meniscal cartilage samples in this group demonstrated histological evidence of chronic rejection, including fibroplasia, encapsulation, mononuclear infiltrates, foreign body giant cells, and eosinophilic infiltrates. There was no difference between the response seen in untreated tissues and that seen in tissues treated with UV irradiation or ozone oxidation. In group 2, the menisci explanted after 1 month displayed extensive infiltration of eosinophils alone or eosinophils mixed with mononuclear cells. The mononuclear infiltrates consisted primarily of CD4+ and CD8+ T cells and of macrophages. The articular cartilage plugs demonstrated only a small area of fibrous encapsulation and leukocyte infiltration at the periphery. This study suggests that xenograft cartilage tissue does not appear to be immunoprivileged and is unsuitable for human implantation due to a chronic rejection mechanism, which is evident already within 1 month after transplantation. In addition, this study may serve as a general model for the primate immune response against xenografts in the absence of hyperacute rejection.

Upper airway closure: a primary source of difficult ventilation with sufentanil induction of anesthesia.

Large-dose opioid induction of anesthesia can lead to difficult ventilation via a mask. Poor ventilatory compliance (VC) may be secondary to "rigid" chest and abdominal wall musculature, glottic closure, or upper airway obstruction. This double-blind study assessed the contribution of the upper airway to poor VC by inducing sufentanil anesthesia in patients undergoing cardiac surgery who are ventilated via a mask (Group M) or endotracheal tube fiberoptically inserted (Group E). After induction of anesthesia with sufentanil 3 microgram/kg from time (T) = 0 min to T = 2 in Group M (n = 17) or Group E(n = 23), VC and adductor pollicis (AP) twitch tension was measured continuously. Immediately prior to muscle relaxant (pipecuronium or doxacurium) administration at T = 3, Group E demonstrated significantly better VC (46 mL/cm H2O [39-55 interquartile range (IQR)]) than Group M (19 mL/cm H2O [7-24 IQR]). The effect of muscle relaxant administration on VC preceded its effect at the AP. After complete relaxation of the AP at T = 9, both groups had similar VC. Difficult ventilation during sufentanil induction of anesthesia lies at the level of the glottis or above. Bypassing these structures with an endotracheal tube overcomes the usual decreased VC.

Human T-cell leukemia virus type I tax/rex DNA and RNA in cutaneous T-cell lymphoma.

Peripheral blood mononuclear cells (PBMCs) and T-cell lines from patients with Sezary syndrome (SS) and skin lesions from patients with mycosis fungoides (MF) were examined by polymerase chain reaction (PCR) for DNA sequences homologous to the human retroviruses human T-lymphotropic virus (HTLV)-I and -II. Results obtained using primers and probes from the tax/rex region of HTLV-I indicate that 72% (18/25) of SS patients PBMCs, 80% (20/25) of T-cell lines established from SS-PBMC, and 30% (3/10) of skin lesions from MF patients were positive for HTLV-I tax/rex region DNA. Sequence analysis of the 127-bp fragment amplified by the tax/rex primers from 4 of these individuals was found to be identical to that in prototypic HTLV-I. Negative results were obtained using primers and probes from the HTLV-I gag region and the HTLV-II gag and tax regions. No PCR products were obtained using all primers and probes using DNA from 9 healthy blood donors and 10 cord bloods. Expression of HTLV-I tax/rex mRNA was found in 4 of 8 Sezary patients, as determined by RNA-PCR, indicating that this viral region is being transcribed in vivo. Exposure to Tax/Rex protein in SS-patients is supported by the fact that serum antibodies against p27rex and p40tax was observed in 43% and 29% of these SS patients, respectively. Although the causal relationship between the HTLV-I tax/rex region and cutaneous T-cell lymphoma (CTCL) remains unclear, these findings support the presence of a truncated HTLV-I retrovirus in CTCL patients.

Sézary T-cell-activating factor induces functional interleukin 2 receptors on T-cells derived from patients with Sézary syndrome.

Sézary syndrome is the leukemic form of cutaneous T-cell lymphoma characterized by circulating neoplastic CD4+ T-cells. Although peripheral blood mononuclear cells from patients with Sézary syndrome have been shown to respond poorly to mitogens, we found that mitogen-activated peripheral blood mononuclear cells from four of five patients with Sézary syndrome produce a 28 kDa protein termed Sézary T-cell activating factor (SAF). SAF renders nonproliferating "resting" T-cells from leukemic patients or healthy donors responsive to interleukin 2 in the absence of a costimulator. We demonstrate that SAF induces functional, high-affinity interleukin 2 receptors on T-cells from Sézary syndrome patients and provide evidence that SAF may be a novel cytokine.

Ventilatory compliance after three sufentanil-pancuronium induction sequences.

Poor ventilatory compliance, a predictable side effect of high-dose opioid induction techniques, is purportedly blunted by pretreatment with nondepolarizing muscle relaxant. This study used both total compliance and a subjective compliance score to compare three different sequences of opioid induction using a 2-min infusion of sufentanil 3 micrograms.kg-1. Nineteen patients in each of three groups received a total of 100 micrograms.kg-1 of pancuronium, in the following randomized double-blinded fashion: control, all pancuronium 1 min after sufentanil; pretreated, 1 mg pancuronium 1 min before sufentanil and the balance of pancuronium 1 min after sufentanil; and mixed, all pancuronium mixed with sufentanil. Topical lidocaine prior to induction permitted early oral airway insertion midway through the sufentanil infusion. Immediately at the conclusion of sufentanil infusion, a tightly fitted mask, anterior jaw thrust, and mechanical ventilator permitted measurement of plateau airway pressure and exhaled volume in five replicates. Pressure and volume measurements were repeated 5 min later. Total compliance was calculated as the median plateau airway pressure divided into its associated exhaled volume. Groups did not differ in demographics. In one control patient and two pretreated patients hemoglobin oxygen saturation as measured by pulse oximetry decreased below 90%. Immediately after sufentanil infusion, the total compliance for control patients of 4.1 ml.cmH2O-1 (mean [2.6-6.5, 95% confidence interval] ) did not differ from that of the pretreated group (6.3 [3.5-11.4] ml.cmH2O-1), but the mixed group exhibited higher compliance (40.3 [33.8-47.9] ml.cmH2O-1) than the other groups (P less than 10(-8]. All groups achieved similar total compliances several minutes after a total of 100 micrograms.kg-1 pancuronium had been administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Malignant and nonmalignant T cell lines from human T cell lymphotropic virus type I-negative patients with Sézary syndrome.

The establishment of an in vitro model for cutaneous T cell lymphomas and Sézary syndrome has been difficult since T cells from individuals with these diseases do not proliferate in response to T cell mitogens. We found that conditioned media, collected from mitogen-activated PBMC from Sézary patients, contain an IL-2 receptor inducing factor. Despite their ostensible proliferative disorder, using a combination Sézary cell-conditioned media and rIL-2, we established IL-2 responsive, human T cell lymphotropic virus type I negative T cell lines from 23 patients, nine of which contain cells with the structural and/or genetic characteristics of neoplastic Sézary T cells.

A clonal CD4-positive T-cell line established from the blood of a patient with Sézary syndrome.

The reported inability to establish long-term T-cell lines from the blood of cutaneous T-cell lymphoma patients with circulating neoplastic T cells has hindered the development of an in vitro system to investigate Sézary syndrome. We have established a rapidly proliferating T-cell line from the peripheral blood of a patient with Sézary syndrome, which expresses a mature helper T-cell phenotype and contains cytogenetic abnormalities and T-cell receptor gene rearrangements identical to those in the patient's blood. The method of establishment and characteristics of this line are described.

The effects of antioxidants on the content of polyunsaturated fatty acids in the hen's egg.

In experiments to see whether, in the possible interests of human health, the polyunsaturated fatty acid (PUFA) content of the chicken's egg can be increased by nutritional means, three strains of hen, light, medium, and heavy, each at the peak of lay, were first fed a basal, commercial, low-fat diet. The hens were then transferred to one of the following diets: basal + safflower oil (SO); basal + SO + butylated hydroxytoluene; or basal + SO + dl-a-toco-pheryl acetate. The diets were designated "Blank", "BHT", and "Vitamin E", respectively, the second and third containing the added antioxidants. The eggs produced were weighed, and their yolks weighed and analysed for lipid components. Additional of SO (7.5%) to the basal diet led to the PUFA content of the yolk lipids rising by 15.4% (linoleic acid, 14.1%), the magnitude of the increases being unaffected by the antioxidants. Diet "BHT" produced larger eggs and yolks than the other diets, but the proportion of yolk was the same on the three types of feed. The total cholesterol content of egg yolks was significantly affected neither by diet, nor by strain or age of hen. The implications of these results are discussed.

The need for co-ordination of research in nutrition.

On two assumptions, first that the number of dietarily essential nutrients for Man and the common monogastric mammals is 40 and, second, that the effects of at least two dietary levels of each nutrient would have to be examined in order to cover the possible nutrient interactions, then 2(40), or approximately 10(12) diets would need to be prepared. This number is beyond human capacity within any reasonable period of time: moreover, it assumes constancy of the non-essential part of the diet. Very few of the possible nutrient interactions have been studied and, as a supplement to piecemeal studies by individual workers, a co-ordinated research programme is desirable. It is noted that, while considerable attention has been paid to the nutrition of growth, the adult stage has been very neglected: the status of adult man in particular calls for attention. Factors concerned in the planning of co-ordinated research are briefly discussed.