Herpes virus infected spermatozoa following density gradient centrifugation for IVF purposes.

Studies have documented the presence of herpes viruses in semen. The aim of our study was to determine whether they persist in semen samples following two-density gradient centrifugation for IVF purposes. Semen samples were collected from 109 men seeking fertility evaluation, prior to IVF treatment. Routine semen analysis was performed according to WHO guidelines. Each sample was treated in a two-density gradient centrifugation using PureSperm (PS). Both untreated and treated samples were screened for the presence of herpes viruses, using PCR. Kruskal-Wallis, chi-square and binomial statistical tests were used; P ≤ 0.05 was considered statistically significant. No statistically significant associations were observed between semen parameters and viral presence. Viral DNA was detected in 54% of semen samples: HSV1/2 in 32 samples, EBV in 49, CMV in 47, HHV6 in 9, HHV7 in 4 and VZV in none. PS gradient failed to remove CMV in 89.36%, HSV1/2 in 59.38% and EBV in 22.45% of samples, while HHV6 and 7 were completely removed. Especially HSV1/2 and CMV seem to persist even following PS treatment. These observations indicate the possible risk of oocyte becoming infected during insemination, by IVF or intracytoplasmic sperm injection, with unknown sequelae. Further studies are required to determine whether any correlation exists between their presence, implantation rate and the outcome of pregnancy.

Detection of herpes virus DNA in post-operative thyroid tissue specimens of patients with autoimmune thyroid disease.

AIM: To demonstrate any differences in the detection of herpes simplex virus type 1 and 2, cytomegalovirus, human herpes virus type 6 and 7 DNA from thyroid tissue blocks of patients with autoimmune thyroid disease and multi-nodular goiter and to propose few mechanisms, which could explain the possible role of herpesvirus infection in the development of thyroid autoimmune responses. MATERIAL-METHODS: Thyroid tissue specimens were obtained postoperatively from 4 patients with multinodular goiter and 18 patients with autoimmune thyroid disease (Graves' disease and Hashimoto thyroiditis). Herpes virus DNA was detected using polymerase chain reaction based assays. RESULTS: No statistically significant differences were observed between autoimmune thyroid disease and multinodular goiter tissue specimens concerning herpes simplex virus type 1, 2 DNA isolation (44.4% vs 0%, P=0.094), human herpes virus type 6 DNA isolation (11.1% vs 0%, P=0.48), human herpes virus type 7 DNA isolation (33.3% vs 25%, P=0.75). No CMV DNA was isolated from any tissue specimen. At least one kind of herpes virus DNA was detected in 13 out of 18 (72.22%) AITD tissue specimens and in 1 out of 4 (25%) MNG tissue specimens (P=0.01). CONCLUSIONS: Although no data are available relating the direct effect of herpes infection on thyroid epithelial cells, a better understanding of how an aberrant immune response against the thyroid gland is initiated and propagated through herpes virus infection is required. Elucidation of the underlying mechanisms may allow the development of new etiologically based therapeutic modalities.

Altered immunophenotypic parameters in infertile women. Possible role of herpes viremia.

PROBLEM: Purpose of this study was to reveal any alteration in peripheral blood lymphocytic concentrations of a large cohort of infertile women and to investigate the possible role of herpes viremia in the peripheral immunostimulation. METHOD OF STUDY: The immunophenotypic characteristics and the presence of herpes viruses DNA in the peripheral blood of 168 infertile women were studied. RESULTS: Peripheral CD56+/CD16+ natural killer (NK) cell concentration, CD56+/CD16- NK cell concentration, white blood cell (WBC) concentration and lymphocyte concentration were statistically correlated to herpes viremia. Epstein-Barr virus (EBV) viremia is related with a limited reduction of CD56+/CD16- cell levels in the peripheral blood of infertile women with regard to the rest of herpes viruses. High T-lymphocyte concentration, CD4+ T-cell concentration and CD8+ T-cell concentration was observed in women positive for three different kinds of herpes viruses (triple viremia) in the peripheral blood. CONCLUSIONS: Assuming that all women under study remained asymptomatic, these data suggest that subclinical herpesvirus viremia may be an important cause of peripheral immunostimulation in women with a history of infertility.

Expression of HLA-DR is reduced in tumor infiltrating immune cells (TIICs) and regional lymph nodes of non-small-cell lung carcinomas. A putative mechanism of tumor-induced immunosuppression?

BACKGROUND: Deregulation of MHC class II molecules consists of a favorable mechanism of tumor evasion from immune surveillance. Among these molecules, HLA-DR antigens are the predominant ones in cancer. In the present study we sought to investigate the ability of tumor infiltrating immune cells (TIICs) to express HLA-DR antigen in the primary tumor site and reactive regional lymph nodes (LNs) in non small cell lung cancer (NSCLC). MATERIALS AND METHODS: Material consisting of 60 NSCLCs with corresponding regional LNs was studied by immunohistochemistry for human leukocyte antigen D-region related (HLA-DR) expression. Control reactive LNs, regional to several different malignant and non-malignant disorders, were also included in the study. RESULTS: Primary tumor site investigation revealed positive HLA-DR cancer cells in 22% of cases, whereas TIICs rarely expressed HLA-DR antigens. The lack of HLA-DR expression in TIICs was gradually attenuated as the distance from the primary tumor site decreased. Regional LN investigation showed that all follicles (paracapsular and deep cortical ones) were HLA-DR-negative in 60% of the LNs; in the remaining 40%, the paracapsular follicles remained negative, while all deep cortical ones were positive. Interestingly, LNs possessing only HLA-DR-negative follicles were more proximal to the primary tumor site compared to those that had only the paracapsular follicles negative. All control reactive LNs, regional to several distinct malignant and non-malignant disorders, were found to be HLA-DR-positive. CONCLUSION: The impairment of HLA-DR expression, detected both in neoplastic and by-stander immune cells, may justify the immunosuppression observed in NSCLC. This phenomenon may be due to a putative soluble factor in the tumor environment secreted by cancer cells.

Characterization of interleukin 2 receptors on B-cell chronic lymphocytic leukemia cells.

The expression and function of IL-2R chains expressed on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed. IL-2R alpha was expressed in 31 out of 34 studied cases; in 17 cases more than 50% of the cells were positive whereas in three cases the proportion of IL-2R alpha+ cells was less than 10%. In two patients, 6 and 13% of the cells were IL-2R beta+, in six other cases only 2-3% of the B-CLL cells could be stained with the TU-27 moAb whereas in all other cases no positive cells could be detected. Equilibrium binding experiments using 125I-rIL2 revealed high (seven out of 15 studied cases), intermediate (four out of 15 cases) and low (five out of 15 cases) affinity IL-2R. The number of high and intermediate affinity IL-2R was low (range: 145-800 and 40-2800 binding sites/cells, respectively). In all cases investigated, both IL-2R alpha and IL-2R beta chain mRNA could be detected, although their quantity was variable from patient to patient. Exogenous recombinant IL-2 induced, in a dose-response manner, cell proliferation in ten out of 23 cases and this effect was independent of the expression of IL-2R alpha; however, only cells expressing high affinity IL-2R could respond to exogenous rIL-2. Moreover, anti-IL-2R alpha moAb could inhibit both spontaneous (in three out of five cases) and IL-2-induced (in five out of five cases) B-CLL cell proliferation. These findings demonstrate that in a subgroup of B-CLL, leukemic cells are dependent on the IL-2/IL-2R system whereas in another group, although cells expressed functional IL-2 binding sites, they could not respond to the mitogenic signal of IL-2.

Monocyte disorders associated with T cell defects in patients with solid tumors.

T cells proliferate in response to autologous monocytes in the autologous mixed lymphocyte reaction (AMLR). AMLR was found to be impaired in patients with advanced cancer (stages III and IV), whereas normal values were found in the early stages of the disease (stages I and II). Peripheral T lymphocytes from patients with advanced stages also exhibited a decreased ability to produce Interleukin-2 (IL-2) during an AMLR response, whereas production of IL-2 by T cells in stages I and II was comparable to that of normal donors. The impaired IL-2 production by T lymphocytes in the AMLR was associated with high concentrations of soluble interleukin-2 receptor (sIL-2R) in culture supernatants and reduced expression of membrane-bound interleukin-2 receptors (IL-2R) on the same AMLR-activated T lymphocytes. These abnormalities in T cells from cancer patients were demonstrated to be associated with dysfunctions of autologous monocytes. Thus monocytes from patients with advanced cancer exhibited diminished expression of HLA-DR antigens and produced low levels of Interleukin-1 beta (IL-1 beta) and Tumor Necrosis Factor a (TNFa). No changes were detected in the expression of HLA-A, -B, -C antigens. The results presented here demonstrate that decreased in vitro T cell responses may be attributed to monocyte dysfunctions in these patients and provide new information for a better understanding of the impaired T cell function in cancer patients.

Biological and biochemical characterization of a factor produced spontaneously by adherent cells of human immunodeficiency virus-infected patients inhibiting interleukin-2 receptor alpha chain (Tac) expression on normal T cells.

Adherent cells from human immunodeficiency virus (HIV)-infected subjects but not from normal blood donors, patients with Gram-positive or -negative bacteremia, active tuberculosis, toxoplasmosis, pulmonary aspergillosis, and cytomegalovirus infection produce spontaneously an activity which inhibits alpha chain of interleukin-2 (Tac) expression and interleukin 2 (IL-2) production by normal activated T cells and IL-2 production by these cells. A similar biologic activity was detected in culture supernatants of in vitro HIV-I-infected normal adherent and leukemic U937 cells. Tac-inhibitory activity is not cytotoxic and it could be detected in serum-free conditioned media. Recombinant granulocyte/macrophage colony-stimulating factor and phorbol myristate acetate stimulation of patients' and normal adherent cells did not enhance specifically the production of the Tac inhibitor. Biologically active conditioned media did not contain infectious virus as well as secreted p24, gp120 viral proteins; the biologic activity could not be abolished by anti-p24, anti-gp120, and anti-nef monoclonal antibodies or human purified polyclonal anti-HIV IgG. Gel filtration of conditioned media followed by anion exchange chromatography resulted in a 1,200-fold degree of purification and revealed that the biologically active molecule was cationic. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of this fraction and gel elution of the proteins showed that the biologic activity was associated with a 29-kD protein which was distinct from alpha- or gamma-interferon, tumor necrosis factor-alpha, and prostaglandin E2. The above findings demonstrate the production of inhibitory factor(s) during HIV infection, which might be involved in the pathogenesis of the patients' immune defect.

Production of a tac inhibitory activity by adherent cells of HIV-infected subjects at different clinical stages.

Studying the mechanisms of the impaired T-cell colony growth from HIV-infected subjects, we have demonstrated that depletion of adherent cells from some patients' peripheral blood mononuclear cells enhanced the plating efficiency of T colony-forming cells. We report here that media conditioned by patients' cells but not normal adherent cells could inhibit the expression of the interleukin-2 receptor alpha (IL-2R alpha) chain but not the IL-2R beta chain in a dose-dependent manner. This inhibitory activity was produced by macrophage-monocyte cells since they displayed the My9+ My7+ OKM1+ phenotype and since adherent cell depletion by complement-mediated cytotoxicity with the My9 monoclonal antibody completely abrogated production of the inhibitory activity. A similar inhibitory activity, which could not be recognized by anti-p24 or anti-gp 120 monoclonal antibody or purified human anti-HIV immun]gobulin G in Western blot assays, could also be detected in culture supernatants of in vitro HIV-infected normal adherent and U937 leukemic cells. Production of IL-2R alpha chain inhibitory activity was associated with a decreased mitogen-induced expression of IL-2R alpha chain on patients' PBMC in 8 of 10 studied cases. Its production could be detected in 82, 58, and 91% of media conditioned by adherent cells from stage II, III, and IV patients, respectively. The amount of IL-2R alpha chain inhibitor released by patients' adherent cells increased during the deterioration of the patients' clinical status, and zidovudine treatment completely abrogated its production in all patients. These findings strongly suggest that production of IL-2R alpha chain inhibitory activity is involved in the pathophysiology of the impaired T-cell responses during HIV infection and could be of clinical relevance during the patients' follow-up.