Use of interleukin 15 to enhance interferon-gamma production by antigen-specific stimulated lymphocytes from rhesus macaques

The enzyme-linked immune spot (ELISPOT) assay is receiving increased attention as a means for quantifying antigen-specific CD8 T-cell responses in rhesus macaques. Further improving the sensitivity of this assay could aid in the evaluation of vaccine candidates and/or immune therapeutic candidates. Interleukin (IL)-15 has been demonstrated to stimulate expansion of human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) and to regulate homeostatic proliferation of CD8+ memory cells. We evaluated the in vitro effect of IL-15 to increase the detection of interferon-gamma (IFN-gamma) production by antigen-specific stimulated lymphocytes from a group of rhesus macaques exposed to simian-human immunodeficiency virus (SHIV) and a second group infected with SIVmac251, before and after antiretroviral treatment (ART). Results from these studies demonstrate that the presence of IL-15 during stimulation in a peptide-based ELISPOT assay greatly enhanced IFN-gamma production in both SHIV and simian immunodeficiency virus (SIV)-infected macaques. IFN-gamma production was mainly mediated by CD8 lymphocytes. The optimal concentrations of IL-15 that give enhancement of IFN-gamma production to specific antigen, without a significant increase in the spontaneous IFN-gamma release, ranged from 0.5 to 2.5 ng/ml. The mean number of IFN-gamma spots was increased 3.1- to 3.6-fold in response to SIV gag or HIV env peptide pools, respectively, in peripheral blood mononuclear cells (PBMC) from SHIV-infected macaques. Similarly, in SIV-infected macaques, IL-15 increased the mean number of IFN-gamma spots 2.7-fold in response to both SIV gag and env peptide pools. In samples obtained after ART in the same macaques, the increase factor was 2.5 for SIV gag and 1.8 for the env peptide pools. Thus, the sensitivity of the ELISPOT assay can be enhanced by addition of IL-15. This modified assay will be useful for detection of low frequencies of IFN-gamma producing cells in rhesus macaques.     

Association study of a SNAP‐25 microsatellite and attention deficit hyperactivity disorder

Several lines of evidence implicate synaptosomal‐associated protein of 25 kDa (SNAP‐25) in the etiology of attention deficit hyperactivity disorder (ADHD). Most notably, the coloboma mouse mutant, considered to be a good animal model of hyperactivity, has a deletion spanning this gene. Introducing a SNAP‐25 transgene into these animals alleviates hyperlocomotion. We have identified a novel microsatellite repeat in SNAP‐25 located between the 5′UTR and the first coding exon, and tested for association with ADHD. Case‐control analyses suggest there may be a role of this polymorphism in ADHD, with one allele over‐represented in controls and another over‐represented in probands. Within‐family tests of linkage and association confirmed these findings. Further work is needed to ascertain the role of SNAP‐25 in ADHD and assess the functional significance of this polymorphism. © 2002 Wiley‐Liss, Inc.     

Expression of cyclooxygenase-2 in osteosarcoma of bone.

Several studies indicate that cyclooxygenase-2 (COX-2) is overexpressed in human malignancies, where it produces high levels of prostaglandins and contributes to tumor growth. In this study we have analyzed the expression of COX-2 in a series of 48 skeletal osteosarcomas of different subtypes by immunohistochemistry. In addition, we examined the effects of the specific COX-2 inhibitor Celecoxib on the growth of the human osteosarcoma cell line SaOS-2. Immunoreactivity for COX-2 was observed in 39 out of 48 tumors (81.2%), 30 (76.9%) of which showed a moderate or diffuse immunostaining. Considering the group of 42 primary osteosarcomas, COX-2 immunoreactivity was significantly higher in high grade osteosarcomas, where moderate or diffuse expression was detected in 23 out of 32 cases (71.8%), than in low grade osteosarcomas, where moderate or diffuse expression was detected in 2 out of 10 cases (20%) (P = 0.008, Fisher exact test). In addition, low COX-2 expression was always associated with a good response to chemotherapy (5 out of 5 cases), whereas moderate or diffuse COX-2 expression was associated with a good response in 11 out of 20 cases (55%) (P = 0.12, Fisher exact test). In SaOS-2 osteosarcoma cells, which express COX-2, treatment with Celecoxib determined inhibition of cell proliferation and induction of apoptosis. These results indicate that COX-2 is expressed at high levels in high grade osteosarcomas and support the use of COX-2 inhibitors to improve both the tumor response to chemotherapy and the outcome of osteosarcoma patients.     

Replicative multivirus infection with cytomegalovirus, herpes simplex virus 1, and parvovirus B19, and latent Epstein-Barr virus infection in the synovial tissue of a psoriatic arthritis patient.

BACKGROUND: Psoriatic arthropathy occurs as complicating feature in about 5-7% of psoriasis patients. Infectious mechanisms including viral antigens have been suggested by serologic data as CD8 T cellular specifity towards viral epitopes. OBJECTIVE AND RESULTS: We here reported a case of a 32-year-old male psoriatic arthritis patient, where we could demonstrate simultaneous infection with cytomegalovirus (CMV), herpes simplex virus type I (HSV1) and parvovirus B19 (B19), as well as latent Epstein-Barr virus (EBV) infection within the synovial tissue by immunohistochemistry (CMV, parvovirus B19, HSV1, EBV-LMP) and DNA-in situ-hybridization (CMV). Serologic examination revealed positive EBV and parvovirus B19-IgG-antibodies, but no antibody response to HSV1 and CMV. CONCLUSION: This case is of special interest, since replicative viral infections have not yet been demonstrated localised in the psoriatic arthritis synovia. Thus, with particular regard to the limited information of the serologic data and the possible need of immuno suppressive therapy direct synovial testing for viral antigenes may be considered in psoriatic arthritis patients.     

Enhancement of human immunodeficiency virus type 1-DNA vaccine potency through incorporation of T-helper 1 molecular adjuvants

Summary: It is clear that the development of a safe and effective vaccine for human immunodeficiency virus type 1 (HIV‐1) remains a crucial goal for controlling the acquired immunodeficiency syndrome epidemic. At present, it is not clear what arm of the immune response correlates with protection from HIV‐1 infection or disease. Therefore, a strong cellular and humoral immune response will likely be needed to control this infection. Among different vaccine alternatives, DNA vaccines appeared more than a decade ago, demonstrating important qualities of inducing both humoral and cellular immune responses in animal models. However, after several years and various clinical studies in humans, supporting the safety of the HIV‐DNA vaccine strategies, it has become clear that their potency should be improved. One way to modulate and enhance the immune responses induced by a DNA vaccine is by including genetic adjuvants such as cytokines, chemokines, or T‐cell costimulatory molecules as part of the vaccine itself. Particularly, vaccine immunogenicity can be modulated by factors that attract professional antigen‐presenting cells, provide additional costimulation, or enhance the uptake of plasmid DNA. This review focuses on developments in the coadministration of molecular adjuvants for the enhancement of HIV‐1 DNA‐vaccine potency.     

Opsonophagocytosis of fluorescent polystyrene beads coupled to Neisseria meningitidis serogroup A, C, Y, or W135 polysaccharide correlates with serum bactericidal activity

We developed a polysaccharide-specific flow cytometric opsonophagocytic assay (OPA) for the simultaneous measurement of functional antibody to Neisseria meningitidis serogroups A, C, Y, and W135. OPA titers significantly correlated with serum bactericidal assay titers for all serogroups tested (mean r = 0.96; P < 0.001). OPA could be used in meningococcal vaccine evaluation.     

Synergistic and inhibitory interactions in the in vitro control of murine megakaryocyte colony formation

The formation of megakaryocyte colonies in agar cultures of murine bone marrow or spleen cells can be stimulated by the addition of interleukin-3 (IL-3), erythropoietin (EPO), thrombopoietin (TPO), or IL-6. However, greater numbers of colonies developed if combinations of two or more of these stimuli were used, particularly combinations including stem cell factor, with maximal numbers of colonies developing with the combination of stem cell factor plus IL-3 plus EPO. The data indicate that most committed progenitor cells in the megakaryocyte lineage were unusual in that they required stimulation by two or more hematopoietic growth factors. In tests using a range of growth factors, G-CSF was exceptional in that it consistently specifically inhibited megakaryocyte colony formation stimulated by EPO, TPO, or IL-6 but not that stimulated by IL-3. The mechanisms involved in this inhibitory action of G-CSF are unknown, but the inhibitory action could be of relevance for the dose-dependent lowering of platelet levels observed in some subjects injected with G-CSF.     

Rapid intraoperative immunohistochemical evaluation of sentinel lymph nodes for metastatic breast carcinoma.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is an integral part of the surgical management of patients with breast cancer. Rapid immunohistochemistry (RIHC) has the potential to increase detection of metastatic carcinoma at the time of frozen section consultation. The authors assessed the accuracy and turnaround time of a newly developed RIHC method for pancytokeratin (RIHC-CK). METHODS: Sixty-six SLNs from 32 patients with breast carcinoma were examined for metastasis using the Zymed Sentinel Lymph Node Rapid IHC Kit. Intraoperative frozen sections (6 mum) of the SLNs were incubated with Zymed anti-pan-cytokeratin/HRP conjugate, diaminobenzidine (DAB), and stained with hematoxylin. Slides were ready within 8 minutes and were interpreted as positive or negative for metastatic carcinoma. Results were compared with previous intraoperative touch preparations, frozen sections, hematoxylin and eosin (Perm H&E), and AEl/3-immunostained permanent sections (Perm CK). RESULTS: Fourteen lymph nodes (19%) in 13 patients tested positive for metastatic carcinoma in Perm H&E, the gold standard. RIHC-CK had the highest sensitivity (92%) of the intraoperative tests, compared with touch preparations (64%) and frozen sections (80%). RIHC-CK showed 94% accuracy, compared with 96% (frozen section) and 93% (touch preparation). The RIHC technique took 8 minutes and was easy to perform and interpret. CONCLUSIONS: Zymed RIHC is a sensitive method for detecting breast cancer metastases in SLNs. The speed, accuracy, and ease of interpretation of the test allow for recognition of micrometastases (<2 mm) that might otherwise be undetectable by current methods of intraoperative evaluation. The prognostic significance and effect on surgical management of micrometastases in SLNs have yet to be determined.     

Identification, characterization and biological activity of oxytocin receptor in the developing human penis

Although abnormalities of the male external genitalia (MEG) are a relatively common problem, little is known concerning the molecular mechanisms that finely regulate penile development. We report here the expression of the oxytocin receptor (OTR) gene by real-time RT-PCR in human fetal tissues (11th-12th week of gestation), including the MEG. The developing penis expressed a very high level of OTR mRNA, only a half log(10) unit lower than fetal central nervous system, used as a positive control. The OTR protein is also highly expressed (western, immunohistochemistry and binding studies) and immunolocalized both in the mesenchymal body and in the surrounding blood capillaries, which will later constitute penile trabeculae and sinusoids. Binding studies using [125I]oxytocin antagonist ([125I]OTA) in cultured human fetal penile smooth muscle cells (hfPSMC) revealed the presence of specific OTR with a high capacity and affinity for oxytocin (OT) and for OTA. Increasing concentrations of OT dose-dependently induced intracellular Ca2+ mobilization. Furthermore, OTR mediated an increase in the proliferation and the migration of hfPSMC. In conclusion, we demonstrate that in the developing human MEG, OTR is highly expressed and might be involved in coordinating timely and appropriate proliferation and migration of the penile cells. Thus, OTR might represent an additional target for investigating human fetal MEG organogenesis.