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.      

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.     

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.     

Factors associated with genotype clustering of Mycobacterium tuberculosis isolates in an ethnically diverse region of southern California,United States

Mycobacterium tuberculosis (Mtb) isolates with identical genotypes, found in different patients, are most likely the result of recent transmission. Mtb strains with closely related genotypes, called clonal complexes, are most likely derived from one another. We examined Mtb genotypes from southern California TB patients from 2005 through 2008 to complete the first comprehensive molecular epidemiology analysis of this complicated and ethnically diverse region. Mtb genotypes were characterized with spoligotype and MIRU-12 typing. MIRU-VNTRplus was utilized to assign genotypes to global lineages and complete cluster analyses. Associations between patient characteristics and genotype clustering and clonal complexes were evaluated using logistic regression and frequency analysis. Of 832 Mtb isolates analyzed, 480 (58%) fell into 94 strain clusters. The majority of isolates were identified as being in the EA1 (31%), LAM (17%) and Haarlem (15%) lineages, but 13 different lineages were found in this region. TB patients with clustered isolates were more likely to be homeless (AOR 3.44, 95% CI 1.65, 7.18) and male (AOR 1.57, 95% CI 1.17, 2.10). Of the 480 clustered strains, 388 aggregated into six clonal complexes.Over 45% of reported TB cases were clustered and likely resulted from recent transmission events. Patients with clustered Mtb isolates that were grouped into clonal complexes had unique socio-demographic characteristics. These data suggest that TB is being transmitted in relatively insular community networks defined by race/ethnicity and country of origin. The addition of clonal complex analysis to simple cluster analysis provides important public health insights into the local transmission of TB in ethnically diverse regions with diverse Mtb genotypes.     

Phototherapeutic keratectomy versus mechanical epithelial removal followed by corneal collagen crosslinking for keratoconus

ObjectiveTo compare the visual outcomes of patients with keratoconus treated with either phototherapeutic keratectomy (PTK) or mechanical epithelial removal prior to corneal collagen crosslinking (CXL).DesignComparative study.ParticipantsThe records of 34 patients (34 eyes) who had PTK (17 eyes) or mechanical (17 eyes) epithelial removal prior to CXL for keratoconus were reviewed retrospectively.MethodsCXL was performed by 1 of 3 surgeons (G.M., W.B.J., or K.B.). Of the eyes, 17 had undergone mechanical epithelial removal prior to CXL and were consecutively selected, after matching with the 17 eyes in the PTK group, for the variables of procedure date, average keratometry, and pachymetry. All eyes had central cones. Manifest refraction spherical equivalent, sphere, cylinder, best-corrected distance visual acuity, and pachymetry were measured and compared preoperatively and in follow-up.ResultsThe mean change between the pre- and postoperative manifest refraction spherical equivalent for the PTK and mechanical groups was 1.68 ± 0.80 and 0.26 ± 0.90, respectively (p < 0.05). The mean change between pre- and postoperative cylinder for the PTK and mechanical groups was 0.53 ± 0.28 and 0 ± 0.18, respectively (p < 0.05). The mean number of lines of improvement in the PTK and mechanical groups were 0.33 ± 0.82 and ?0.58 ± 0.45 lines, respectively (p > 0.05).ConclusionsEarly results suggest that CXL with laser epithelial removal is superior to CXL with mechanical epithelial removal because it reduces refractive error in qualified patients. Although not statistically significant, there was also a trend for PTK CXL patients to have better visual outcomes.