Margareta Andersson

Decontamination of leukemic cells and enrichment of germ cells from testicular samples from rats with Roser’s T-cell leukemia by flow cytometric sorting

Testicular germ cell transplantation is a novel strategy for preservation of fertility in prepubertal cancer patients, but the risk of reseeding tumor cells into cured patients presently limits clinical application of this approach. To date, no systematic evaluation of the limitations of surface marker-based decontamination of testicular samples with acute lymphoblastic leukemia has been performed. Here, surface markers for leukemic (CD4 and major histocompatibility complex class I) and germ cells (epithelia cell adhesion molecule) in testicular samples infiltrated with Rosers T-cell leukemia were identified. These markers were then used to delete leukemic cells and/or select for germ cells by flow cytometry (FACS). The resulting cell populations were analyzed by FACS, immunocytochemistry, or evaluation of leukemia transmission in syngeneic piebald variegated rats. Simple positive selection of germ cells or deletion of leukemic cells using specific surface markers was unable to effectively decontaminate testicular samples. The poor specificity of spermatogonial surface markers and aggregation of germ and leukemic cells limited the positive selection of germ cells, while immunophenotypic variation among lymphoblastic leukemia cells prevented adequate deletion of leukemic cells. Enzymatic treatment to disperse the testicular cells and feature of the intratesticular environment contributed to this immunophenotypic variation. Only germ cell selection in combination with leukemic cell deletion prevented leukemia transmission in association with intratesticular injection of the sorted cells. However, with such combined sorting, only 0.23% of the original testicular cells were recovered. With presently available techniques, flow cytometric purification of germ cells from a leukemic donor is not sufficiently effective or safe for clinical use.

Two different mechanisms for modulation of bronchoconstriction in guinea-pigs by cyclooxygenase metabolites☆

Leukotriene D(4) (LTD(4))-induced bronchoconstriction in guinea-pig airways has a cyclooxygenase (COX)-dependent component. The main objective of this study was to establish if prostaglandin (PG) D(2)-induced bronchoconstriction also was modulated by COX products. The effects of non-selective and selective COX-1 and COX-2 inhibitors on bronchoconstriction induced by LTD(4) and PGD(2) were investigated in the perfused and ventilated guinea-pig lung (IPL). Both LTD(4)-induced bronchoconstriction and thromboxane (TX) A(2) release was suppressed by COX inhibitors or by TX synthesis inhibition. The release of additional COX products following CysLT(1) receptor activation by LTD(4) was established by measurements of immunoreactive 6-keto PGF(1alpha) (a stable metabolite of PGI(2)) and PGE(2). In contrast, TP receptor-mediated bronchoconstriction by PGD(2) was somewhat enhanced by COX inhibitors, and there was no measurable release of COX products after TP receptor activation with U-46619. PGE(2) was bronchoprotective in IPL as it inhibited the histamine-induced bronchoconstriction. In the isolated guinea-pig trachea, neither PGD(2) nor U-46619 actively released PGE(2), but continuous production of PGE(2) and PGI(2) was established, and the response to PGD(2) was enhanced also in the trachea by COX inhibition. The study documented that bronchoconstriction induced by LTD(4) and PGD(2) in IPL was modulated differently by COX products. Whereas bronchoconstriction induced by LTD(4) was amplified predominantly by secondarily released TXA(2), that induced by PGD(2) was attenuated by bronchoprotective PGE(2) and PGI(2), presumably tonically produced in the airways.

Expression of the signal transduction molecule ζ in peripheral and tumour‐associated lymphocytes in Hodgkin's disease in relation to the Epstein–Barr virus status of the tumour cells

Summary. We investigated whether the described immune evasion of Epstein–Barr virus (EBV)‐infected malignant Hodgkin and Reed‐Sternberg (HRS) cells in Hodgkins disease (HD) is paralleled by a disturbed expression of the signal transduction molecule ζ associated with CD3 and CD16 in tumour‐associated T lymphocytes (TAL). Flow cytometric analysis revealed a significantly lower ζ expression in CD3+/4+, CD3+/8+ and CD16+ patient peripheral blood lymphocytes (PBL; n = 10) compared with normal donor PBLs (n = 11). When patient PBLs were compared with the corresponding TAL, the latter showed a significantly higher (CD3+/4+) or equal (CD3+/8+) ζ expression. The EBV status of the tumours did not correlate with ζ expression in the TAL. Immunohistochemical staining revealed ζ‐positive lymphocytes among the adjacent bystander cells of the HRS cells in all analysed tumours (n = 8), irrespective of tumour EBV status. In conclusion, these results do not support downregulation of ζ in TAL as a critical mechanism contributing specifically to the immune escape of EBV+ HRS cells.

Setae from Larvae of the Northern Processionary Moth (Thaumetopoea pinivora, TP) Stimulate Proliferation of Human Blood Lymphocytes In Vitro

Larvae of the Northern pine processionary moth (Thaumetopoea pinivora, TP) carry microscopic needles (setae), which by penetrating skin and mucous membranes, may cause inflammatory/immune derived symptoms in man. In the present study the stimulatory effects of setae on human blood lymphocytes in vitro was investigated. Blood mononuclear cells were separated from venous blood or buffy coat of ten healthy individuals, six previously exposed to setae and four with no known exposure. Lymphoproliferation was measured as uptake of 3H-thymidine. Setae were prepared from TP larvae. Setae and saline setae extracts stimulated proliferation of T-lymphocytes in the presence of monocytic cells. Stimulation was pronounced in cells from persons who had been exposed to setae, and weak in cells from non-exposed donors. Chitin also induced lymphocyte proliferation in most donors, but to a lesser extent and independently of donors previous exposure to setae. In conclusion, setae contain molecules that in the presence of monocytes activate human T-lymphocytes to proliferation. The antigenic nature of stimulatory molecules was supported by the significantly stronger lymphocyte response in persons previously exposed to setae than in non-exposed donors. The nature of such molecules remains to be defined.

Ontogenesis of Ap-2γ Expression in Rat Testes

Searching for useful markers of spermatogonial stem cells and their differentiation, we used rat testes from ages representing different stages of testicular maturation to investigate the expression profile of transcription factor activation protein-2γ (Ap-2γ). The immunohistochemical and immunocytochemical evaluation using Ap-2γ and promyelocytic leukemia zinc finger in combination with sorting of CD9 and CD90 positive cells (undifferentiated spermatogonia) by fluorescence-activated cell sorting was performed. Our experiments revealed that Ap-2γ is detectable in testes of late fetal age and up to 60 days postnatally and is expressed in gonocytes and spermatogonia from late fetal age throughout all maturational stages. Restricted nuclear expression of Ap-2γ to undifferentiated male germ cells was verified by coexpression of Ap-2γ with promyelocytic leukemia zinc finger in sections of paraffin-embedded testes as well as in cells sorted positive for CD9 and CD90 expression. Our study demonstrated clearly that nuclear expression of Ap-2γ is a useful marker for identifying undifferentiated male germ cells, although its functional role is yet to be fully explored.