A new combined in-vitro test model for the identification of substances affecting essential sperm functions [published erratum appears in Hum Reprod 1997 Nov;12(11):2580]

Binding of mammalian spermatozoa to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. It has been postulated that xenobiotics that are released in the environment as well as exposure to pharmaceutical medications may be associated with reproductive problems in men and wildlife. Examining physiological and non-physiological effects of particular compounds on sperm functions requires high quality in-vitro test systems. We established a reliable combined in-vitro test system with bovine gametes and evaluated if aliquots of pooled post-thaw spermatozoa are suitable for examining essential sperm functions. Using cryopreserved semen, the PSA-FITC/Hoechst 33258 staining procedure was applicable to evaluate the acrosomal status and cell viability. In the bovine hemizona assay, hemizona indices revealed no differences between cryopreserved and fresh semen. Treatment of post-thaw bovine spermatozoa with progesterone (1 microM or bovine follicular fluid (20%) induced the acrosome reaction from 12% (untreated spermatozoa) to 25% (P < 0.001) and to 22% [corrected] (P < 0.01), respectively. Incubation of both compounds (1 microM progesterone and 20% follicular fluid) raised the percentage of acrosome-reacted spermatozoa to 30% (P < 0001). Our results demonstrate that cryopreserved semen can be integrated into an in-vitro screening model for reproductive toxicology testing. Pooled, cryopreserved bovine spermatozoa will thus permit reproducible experiments for clinical and basic science purposes and may also be applicable for the human system.      

Cloning, functional activities and in vivo tissue distribution of rat NKR-P1+ TCR alpha beta + cells

We have successfully cloned nine NKR-P1+ TCR alpha beta + cells from PVG rat spleens, utilizing murine macrophage inflammatory protein-1 alpha (MIP-1 alpha) and IL-2. These clones are either double negative (DN, CD4-CD8-), which included clones 3.31, 3.71, 4.19, 4.59 and 4.65, or single positive (SP, CD4+CD8-), which included clones 1.64, 3.8, 3.76 and 3.78. No CD8+ clone was recovered. All nine clones are restricted in terms of their expression of the V beta antigens, since they express V beta 8.2 but not V beta 8.5, V beta 10 or V beta 16. These clones are agranular and they fall to generate NK or LAK activity upon incubation with IL-2, IL-12 or their combination. On the basis of their production of intracellular cytokines they can be divided into three categories: (I) SP clones (1.64, 3.8, 3.76 and 3.78) do not produce IL-2 or IL-4, but produce IFN-gamma and IL-12, and they vary in their production of IL-1, RANTES or tumor necrosis factor (TNF)-alpha; (II) DN clones 4.59 and 4.65 produce IL-1 alpha and IFN-gamma only, and fall to produce other cytokines; and (III) DN clones 3.31, 3.71 and 4.19 produce IL-1 alpha, IL-1 beta, IL-2, IL-12, IFN-gamma, RANTES and TNF-alpha. From all the clones examined only DN clones 3.31 and to a lesser degree 4.19 produce IL-4. In vivo tissue localization of clones 3.8, 3.31 and 4.59 shows that these cells distribute into the liver and bone marrow 24 h post i.v. administration. Their accumulation in the liver and bone marrow along with their ability to secrete various cytokines suggest that these cells may influence the generation, differentiation or apoptosis of immune or hematopoietic cells.      

Pediatric peripheral blood progenitor cell collection: haemonetics MCS 3P versus COBE Spectra versus Fresenius AS104

BACKGROUND: An increasing number of apheresis machines are becoming available for peripheral blood progenitor cell (PBPC) collection in children. STUDY DESIGN AND METHODS: At the Children's Hospital of Florence (Italy), three apheresis machines were evaluated: MCS 3P (Haemonetics) (10 procedures in 4 patients, aged 10–12 years, weight 23.5-64 kg), Spectra, (COBE) (8 procedures in 3 patients, aged 4–17 years, weight 19–59 kg), and AS104 (Fresenius) (24 procedures in 9 patients, aged 2–16 years, weight 13.6-60 kg). For PBPC quantitative analysis, CD34 cytofluorimetry was employed. Relevant variables analyzed included efficiency of CD34+ cell extraction and enrichment, mononuclear cell purity and red cell contamination of the apheresis components, and platelet count decreases after leukapheresis. RESULTS: No significant differences in CD34+ cell-extraction abilities were found. However, the AS104 provided consistently purer leukapheresis components in terms of mononuclear cell and CD34+ cell enrichment (441 +/− 59%, vs. 240 +/− 35% and 290 +/− 42% for MCS 3P and Spectra, respectively). Postapheresis platelet counts dropped the least with the AS104. The smallest patient who underwent apheresis with MCS 3P (the only machine working on discontinuous flow and hence with greater volume shifts) weighed 23.5 kg and tolerated the procedure well, with no signs of hemodynamic instability. No significant complications were observed. CONCLUSION: All machines seem to have comparable PBPC extraction efficiency, but the AS104 seems to give the component with the greatest PBPC enrichment. This feature might be relevant for further ex vivo cell processing (CD34+ cell selection, expansion, and so on).     

Stimulation of eosinophil production in vitro by eosinophilopoietin and spleen-cell-derived eosinophil growth-stimulating factor

Eosinophilopoietin (EPP) was previously characterized by the ability to stimulate eosinophil production in vivo, but these studies could not ascertain whether EPP had a direct effect on the bone marrow or acted indirectly by causing release of eosinophilopoietic activity by other tissues. The present studies demonstrate that EPP stimulates eosinophil growth in liquid culture of mouse bone marrow in vitro. The timing of stimulation by EPP in vivo and in vitro were parallel, with maximal eosinophil growth after 48 hr. Moreover, EPP appears similar to, and possible identical with, the eosinophil growth-stimulating substance (EO-GSF) released by antigenic stimulation of immune nonadherent spleen cells. Both EPP and EO-GSF are of low molecular weight, both produce stimulation of eosinophil growth with identical kinetics, and both produced similar dose-response curves in the liquid culture system.     

Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques [see comments]

Although peripheral blood stem cell collections (PBSC) are thought to have less tumor involvement than bone marrow (BM), the incidence of circulating tumor cells in patients with breast cancer has not been widely investigated. We prospectively investigated the incidence and viability of tumor cell involvement in PBSC and BM collections from breast cancer patients undergoing high-dose chemotherapy/hematopoietic stem cell transplantation. Paired samples of PBSC and BM from 48 patients were analyzed using an immunocytochemical technique that detects one epithelial-derived tumor cell per 5 x 10(5) mononuclear cells. Immunostained tumor cells were detected in 9.8% (13/133) PBSC specimens from 9/48 (18.7%) patients and in 62.3% (38/61) BM specimens from 32/48 (66.7%) patients, a significantly higher rate than in PBSC (P < .005). The geometric mean concentration of tumor cells in contaminated PBSC specimens was 0.8/10(5) mononuclear cells (range 0.33 to 2.0/10(5)) compared with 22.9/10(5) mononuclear cells in BM (range 1 to 3,000/10(5), P < .0001). In culture experiments, clonogenic tumor colonies grew in 21/26 immunocytochemically positive specimens. No tumor colony growth was detected in 30/32 immunocytochemically negative specimens. Immunocytochemical detection of tumor involvement in BM and PBSC correlated significantly with in vitro clonogenic growth (P < .0001). We conclude that PBSC contain fewer tumor cells than paired BM specimens from patients with advanced breast cancer and that these tumor cells appear to be capable of clonogenic growth in vitro.     

Pancreatic undifferentiated carcinoma with osteoclast‐like giant cells is genetically similar to,but clinically distinct from,conventional ductal adenocarcinoma

Undifferentiated carcinoma of the pancreas with osteoclast‐like giant cells (UCOGC) is currently considered a morphologically and clinically distinct variant of pancreatic ductal adenocarcinoma (PDAC). In this study, we report clinical and pathological features of a series of 22 UCOGCs, including the whole exome sequencing of eight UCOGCs. We observed that 60% of the UCOGCs contained a well‐defined epithelial component and that patients with pure UCOGC had a significantly better prognosis than did those with an UCOGC with an associated epithelial neoplasm. The genetic alterations in UCOGC are strikingly similar to those known to drive conventional PDAC, including activating mutations in the oncogene KRAS and inactivating mutations in the tumor suppressor genes CDKN2A, TP53, and SMAD4. These results further support the classification of UCOGC as a PDAC variant and suggest that somatic mutations are not the determinants of the unique phenotype of UCOGC. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.