Ranjit S. Parhar

Prediction of successful embryo implantation by measuring interleukin-1-alpha and immunosuppressive factor(s) in preimplantation embryo culture fluid*

To find a better predictor of pregnancy after in vitro fertilization (IVF), supernatant fluids from embryo culture media were analyzed after 24 hours and 48 hours for the presence of interleukin-1-alpha (IL-1), interleukin-2, and the percent of immunosuppression. The measurements were performed on 108 consecutive IVF cycles between June 1989 and October 1989. The IL-1 level +/- SD in the 24-hour aliquots of the supernatant of embryo culture fluid was 66.2 +/- 10.2 pg/mL in all viable pregnancy cycles and 35.4 +/- 9.01 pg/mL in unsuccessful cycles. The percent of immunosuppression after 24 hours was 22.06% +/- 4.5% in viable pregnancy cycles and 7.3 +/- 5.5% in unsuccessful cycles. The percent of immunosuppression 48 hours after ovum pick-up was generally decreased in all embryo culture fluid, showing 17.5% +/- 4.4% in viable pregnancy cycles and 3.8% +/- 3.6% in unsuccessful cycles. Interleukin-1 levels in the 48-hour aliquots were moderately decreased, being 39.0 +/- 6.3 pg/mL in viable pregnancy cycles and 34.3 +/- 4.7 pg/mL in the unsuccessful cycles. In 24 hours, embryo culture aliquots IL-1 level greater than 60 pg/mL was seen in 17 of 21 (80.9%) pregnancy cycles, and the combined data of IL-1 level greater than 60 pg/mL and/or greater than 20 percent of immunosuppression predicted 21 of 21 (100%) pregnancy cycles.

Alpha-gal-independent dual recognition and activation of xenogeneic endothelial cells and human naïve natural killer cells

Background. Interaction between vascularized xeno-graft and host immune system is thought to occur via Galactose &agr; (1,3) Galactose (Gal&agr; 1,3 gal) structures decorating the xenograft. Methods. We raised anti-Gal&agr; 1,3 gal-BSA polyclonal antibodies in baboons and investigated effect(s) of these antibodies as well as soluble Gal&agr; 1,3 gal-BSA on human naïve natural killer (NK) cell interactions with porcine aortic endothelial cells. Results. We demonstrate that human naïve (unstimulated) NK cells recognize xenogeneic endothelial cells under conditions where binding to the Gal&agr; 1,3 gal structures is minimized by the presence of blocking anti-Gal&agr; 1,3 gal IgG or soluble Gal&agr; 1–3 gal and in the absence of xenoreactive natural antibodies and complement. After xenogeneic encounter both endothelial cells and human NK cells are activated. Endothelial cell activation is rapid and is manifested initially by an intraendothelial calcium transient and subsequently by expression of P-selectin and vascular endothelial cell adhesion molecule-1 on the xenoendothelium surface. NK cell activation is manifested by increased expression of perforin and increased cytotoxicity towards the xenoendothelium. Neither recognition nor activation of the xenoendothelium was affected by the introduction of either anti-Gal&agr; 1,3 gal IgG or soluble Gal&agr; 1–3 gal. Conclusion. Our data provide evidence that innate immune cells, such as NK cells, recognize and activate xenoendothelial cells independently of Gal&agr; 1–3 gal structures and raise the possibility of novel interactive sites on both human naïve NK cells and discordant xenogeneic endothelium.

Regulation of fat storage and reproduction by Kruppel-like transcription factor KLF3 and fat-associated genes in Caenorhabditis elegans.

Coordinated regulation of fat storage and utilization is essential for energy homeostasis, and its disruption is associated with metabolic syndrome and atherosclerosis in humans. Across species, Krüppel-like transcription factors (KLFs) have been identified as key components of adipogenesis. In humans, KLF14 acts as a master transregulator of adipose gene expression in type 2 diabetes and cis-acting expression quantitative trait locus associated with high-density lipoprotein cholesterol. Herein we report that, in Caenorhabditis elegans, mutants in klf-3 accumulate large fat droplets rich in neutral lipids in the intestine; this lipid accumulation is associated with an increase in triglyceride levels. The klf-3 mutants show normal pharyngeal pumping; however, they are sterile or semisterile. We explored important genetic interactions of klf-3 with the genes encoding enzymes involved in fatty acid (FA) β-oxidation in mitochondria or peroxisomes and FA synthesis in the cytosol, namely acyl-CoA synthetase (acs-1 and acs-2), acyl-CoA oxidase (F08A8.1 and F08A8.2), and stearoyl-CoA desaturase (fat-7). We show that mutations or RNA interference in these genes increases fat deposits in the intestine of acs-1, acs-2, F08A8.1, and F08A8 animals. We further show that acs-1 and F08A8.1 influence larval development and fertility, respectively. Thus, KLF3 may regulate FA utilization in the intestine and reproductive tissue. We demonstrate that depletion of F08A8.1 activity, but not of acs-1, acs-2, F08A8.2, or fat-7 activity, enhances the fat phenotype of the klf-3 mutant. Taken together, these results suggest that klf-3 regulates lipid metabolism, along with acs-1, acs-2, F08A8.1, and F08A8.2, by promoting FA β-oxidation and, in parallel, may contribute to normal reproductive behavior and fecundity in C. elegans.

Presence of leukemia inhibitory factor and interleukin-12 in human Follicular fluid during Follicular growth

PROBLEM: Cytokines have been shown to be present in human follicular fluid and have regulatory functions on follicular maturation. The presence of leukemia inhibitory factor (LIF) and interleukin (IL)‐12 in human follicular fluid obtained at different stages of maturation was investigated.

Cannabinoid 2 receptor induction by IL-12 and its potential as a therapeutic target for the treatment of anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma is the most aggressive type of thyroid malignancies. Previously, we demonstrated that tumorigenicity of anaplastic thyroid carcinoma cell line ARO was significantly reduced following interleukin (IL)-12 gene transfer. We suspected that tumor target structure in ARO/IL-12 cells might be changed and such a change may make them more susceptible to be killed through mechanisms apart from natural killer-dependent pathway. To identify genes involved, we examined gene expression profile of ARO and ARO/IL-12 by microarray analysis of 3757 genes. The most highly expressed gene was cannabinoid receptor 2 (CB2), which was expressed eightfold higher in ARO/IL-12 cells than ARO cells. CB2 agonist JWH133 and mixed CB1/CB2 agonist WIN-55,212–2 could induce significantly higher rate of apoptosis in ARO/IL-12 than ARO cells. Similar results were obtained when ARO cells were transfected with CB2 transgene (ARO/CB2). A considerable regression of thyroid tumors generated by inoculation of ARO/CB2 cells was observed in nude mice following local administration of JWH133. We also demonstrated significant increase in the induction of apoptosis in ARO/IL12 and ARO/CB2 cells following incubation with 15 nM paclitaxel, indicating that tumor cells were sensitized to chemotherapy. These data suggest that CB2 overexpression may contribute to the regression of human anaplastic thyroid tumor in nude mice following IL-12 gene transfer. Given that cannabinoids have shown antitumor effects in many types of cancer models, CB2 may be a viable therapeutic target for the treatment of anaplastic thyroid carcinoma.

A Krüppel-Like Factor in Caenorhabditis elegans with Essential Roles in Fat Regulation, Cell Death, and Phagocytosis

We demonstrate that a Caenorhabditis elegans Krüppel-like transcription factor is involved in fat regulation, cell death, and phagocytosis in C. elegans. Suppression of C. elegans klf-1 function by RNA interference (RNAi) results in increased fat storage in the intestine of the RNAi worm that directly or indirectly causes germ cells to die. These dead cells are not engulfed or phagocytosed in the RNAi worm. High-level expression of Ce-klf-1 during larval development, as well as its specific localization in the worms intestine, supports a direct role for Ce-klf-1 in fat regulation. The C. elegans klf-1 encodes a C(2)H(2) zinc finger protein that is known to act as transcriptional modulator of tissue-specific expression. Members of the Krüppel-like factor (KLF) family play a variety of important roles in vertebrate tissue differentiation. KLFs have recently been implicated in energy and glucose homeostasis through their expression in pancreas, adipose, liver, and muscle tissues. The extensive fat storage and increased cell death in the Ce-klf-1 RNAi worm is important in that it may explain the connection between Ce-klf-1 signaling, cell death, and fat storage. This is the first evidence involving Ce-KLF-1 protein in such functions. In future studies, a thorough analysis of cellular functions of other members of C. elegans Krüppel-like transcription factors together with their interactions and pathway activities with other molecular partners should yield significant insights into mammalian KLF proteins.

Inhibition of neutrophil functions by human immunoglobulin E

Incubation of human neutrophils with human immunoglobulin (Ig) E caused dose-dependent inhibition of adhesion, phagocytosis, secretion of myeloperoxidase, and oxygen radical production. The concentrations of IgE that caused 50% inhibition of adhesion, phagocytosis, and secretion were 2 +/- 0.3, 2.16 +/- 0.21, and 1.95 +/- 0.28 ng/ml, respectively. Oxidase activation as measured by luminol-dependent chemiluminescence by the receptor-mediated N-formyl-methionyl-leucyl-phenylalanine, phorbol 12-myristate 13-acetate, or the particulate stimulus Staphylococcus aureus was inhibited by IgE with concentrations causing 50% effect of 1.2 +/- 0.13, 1.09 +/- 0.16, and 0.6 +/- 0.09 ng/ml, respectively. IgE also inhibited oxygen consumption rate and cytochrome c reduction with similar K0.5 values. The effect of IgE was unlikely to be due to nonspecific cytotoxicity because trypan blue exclusion test and the cytoplasmic marker lactate dehydrogenase revealed that the cells retained their viability after IgE treatment. Similar or higher concentrations of IgG invoked either no inhibition or a slight enhancement of neutrophil functions. Pretreatment of neutrophils with IgG failed to affect the IgE-induced inhibition. Because the effect of IgE occurs at concentrations less than those reported in hyperimmunoglobulinemia E, we propose that direct inhibition of neutrophil functions may underlie the pathogenesis of recurrent infection associated with hyperimmunoglobulinemia E.

Characterization of antigens and allergens of date palm (Pheonix dactylifera) pollen

Antigenic and allergenic components of date palm (Phoenix dactylifera) pollen were investigated to observe their effects on the skin test reactivity, lymphocyte blastogenesis and cytokine production in atopic and healthy individuals. Date pollen extracts were fractionated using SDS‐PAGE and Sephacryl S‐200 gel filtration. Western blotting of SDS‐PAGE separated components with antiserum raised against whole pollen extract in rabbits revealed at least 22 immunoreactive bands ranging in molecular weight between 12 and 94 kD. The immunogenicity of the pollen extract was further confirmed by strong positive reactions in ELISA and Ouchterlonys double diffusion tests. Immunoblot analyses revealed IgG and IgE reactive components (14‐94 kD for IgG and 12‐90 kD for IgE) in the skin test‐positive patients’ sera against whole pollen extract. Fifteen of 60 atopies reacted positively to either whole or some fractions of date pollen extract when skin tested. In response to whole or components of date pollen extract atopic patients showed differential peripheral blood lymphocyte (PBE) proliferative response and cytokine (IL‐2, IL‐4) production when compared with PBE of normal subjects. Our findings strongly suggest that date palm pollen should be considered a reaginic component and should be included in the battery of allergens for determining the allergic status of atopic patients, particularly in those parts of the world where the date palm is grown commercially.

Human neutrophil gene expression profiling following xenogeneic encounter with porcine aortic endothelial cells: the occult role of neutrophils in xenograft rejection revealed

The role of innate immune cells in the recognition and activation of xenogeneic endothelium has always been considered secondary to the initial insult of xenoreactive natural antibodies (XNA) and complement. It was argued, however, that innate immune cells are capable of recognizing and activating xenogeneic endothelium in the absence XNA and complement. Here, we show that porcine aortic endothelial cells (PAECs) activate human neutrophils directly. This contact‐dependent activation causes a transient calcium rise leading to increased reactive oxygen metabolite (ROM) production. Neutrophil gene‐expression profiling using an adenylate uridylate‐rich element‐based microarray revealed a dramatic change in the neutrophil gene profiles upon exposure to PAECs. The PAEC‐dependent neutrophil transcriptional activity was further confirmed by real‐time polymerase chain reaction, which revealed a rapid increase in the mRNA message of a number of inflammatory cytokines. The activation of human neutrophils by PAECs was independent of galactose α1,3‐galactose (Galα1,3‐gal) structures, as inclusion of saturating concentrations of anti‐Galα1,3‐gal l antibodies had no significant effect. Furthermore, this activation was inhibited in the presence of the calcium chelator 1,2‐bis(O‐aminophenyl‐ethane‐ethane)‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester and the ROM inhibitor diphelylene iodonium. Our data illustrate the direct activation of innate immune cells by PAECs in the absence of XNA and complement and suggest alternative recognition sites between PAECs and human innate immune cells.

Major allergens of date palm (Phoenix dactylifera L.) pollen. Identification of IgE-binding components by ELISA and immunoblot analysis.

The IgE‐binding components of date palm (Phoenix dactylifera L.) pollen were determined by ELISA and Western blotting in atopic patients in order to identify its major allergens. From a pool of previously identified allergenic fractions and sera from 15 skin‐test‐positive, atopic subjects, four components of 12, 14.4, 57, and 65–67 kDa were found to bind IgE in 80–93% of sera. Two other components of molecular masses 28–30 and 37–40 kDa also bound 60–80 % of atopic sera. The immunologic specificity of date‐pollen allergen that induced antibody response in sera of atopic patients was confirmed with ELISA. Furthermore, most of the reactivity in pooled positive atopic serum and antiserum raised in rabbits was eliminated after the sera were absorbed with the allergen. IgG immunoblot analyses showed varying degrees of cross‐reactivity with common local allergens, notably Bermuda grass, but were generally of low intensity. These results indicate that date pollen has six major allergens with the 12, 14.4, 57, and 65–67 kDa bands binding 80–93%, and the 28–30 and 37–40 kDa bands 60–80% of atopic sera. We propose that these major allergens be assigned the notations Pho d I to Pho d VI in the order listed.