Two alternative methods for the retrieval of somatic cell populations from the mouse ovary.

Many modern techniques employed to uncover the molecular fundamentals underlying biological processes require dissociated cells as their starting point/substrate. Investigations into ovarian endocrinology or folliculogenesis, therefore, necessitate robust protocols for dissociating the ovary into its constituent cell populations. While in the mouse, methods to obtain individual, mature follicles are well-established, the separation and isolation of single cells of all types from early mouse follicles, including somatic cells, has been more challenging. Herein we present two methods for the isolation of somatic cells in the ovary. These methods are suitable for a range of applications relating to the study of folliculogenesis and mouse ovarian development. First, an enzymatic dissociation utilising collagenase and a temporary, primary cell culture step using neonatal mouse ovaries which yields large quantities of granulosa cells from primordial, activating, and primary follicles. Second, a rapid papain dissociation resulting in a high viability single cell suspension of ovarian somatic cells in less than an hour, which can be applied from embryonic to adult ovarian samples. Collectively these protocols can be applied to a broad array of investigations with unique advantages and benefits pertaining to both.

Signal transducer and activator of transcription (STAT) 1 and STAT3 are expressed in the human ovary and have Janus kinase 1-independent functions in the COV434 human granulosa cell line.

Ovarian granulosa cells are fundamental for oocyte maintenance and maturation. Recent studies have demonstrated the importance of members of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway in the granulosa cell population of mouse and horse ovaries, with perturbation of JAK1 signalling in the mouse shown to impair oocyte maintenance and accelerate primordial follicle activation. The presence and role of the JAK/STAT pathway in human granulosa cells has yet to be elucidated. In this study, expression of JAK1, STAT1 and STAT3 was detected in oocytes and granulosa cells of human ovarian sections from fetal (40 weeks gestation) and premenopausal ovaries (34-41 years of age; n=3). To determine the effects of JAK1 signalling in granulosa cells, the human granulosa-like cell line COV434 was used, with JAK1 inhibition using ruxolitinib. Chemical inhibition of JAK1 in COV434 cells with 100nM ruxolitinib for 72h resulted in significant increases in STAT3 mRNA (P=0.034) and p-Y701-STAT1 protein (P=0.0117), demonstrating a role for JAK1 in modulating STAT in granulosa cells. This study implicates a conserved role for JAK/STAT signalling in human ovary development, warranting further investigation of this pathway in human granulosa cell function.

Odor tracking flight of male Manduca sexta moths along plumes of different cross-sectional area.

Males of the hawkmoth, Manduca sexta, track wind-borne plumes of female sex pheromone by flying upwind, while continuously turning from side-to-side and changing altitude. Their characteristic "zigzagging" trajectory has long been thought to result from the interaction of two mechanisms, an odor-modulated orientation to wind and a built-in central nervous system turning program. An interesting and as of yet unanswered question about this tracking behavior is how the cross-section of an odor plume or its clean-air "edges" affects moths' odor tracking behavior. This study attempts to address this question by video recording and analyzing the behavior of freely flying M. sexta males tracking plumes from pheromone sources of different lengths and orientations with equal odor concentration per unit area. Our results showed that moths generated significantly wider tracks in wide plumes from the longest horizontally-oriented sources as compared to narrower point-source plumes, but had relatively unaltered tracks when orienting to plumes from the same length sources oriented vertically. This suggests that in addition to wind and the presence of pheromones, the area of the plume's cross section or its edges may also play an important role in the plume tracking mechanisms of M. sexta.

Monocyte infiltration and kidney allograft dysfunction during acute rejection.

Multiple cell types infiltrate acutely rejecting renal allografts. Typically, monocytes and T cells predominate. Although T cells are known to be required for acute rejection, the degree to which monocytes influence this process remains incompletely defined. Specifically, it has not been established to what degree monocytes impact the clinical phenotype of rejection or how their influence compares to that of T cells. We therefore investigated the relative impact of T cells and monocytes by correlating their presence as measured by immunohistochemical staining with the magnitude of the acute change in renal function at the time of biopsy in 78 consecutive patients with histological acute rejection. We found that functional impairment was strongly associated with the degree of overall cellular infiltration as scored using Banff criteria. However, when cell types were considered, monocyte infiltration was quantitatively associated with renal dysfunction while T-cell infiltration was not. Similarly, renal tubular stress, as indicated by HLA-DR expression, increased with monocyte but not T-cell infiltration. These data suggest that acute allograft dysfunction is most closely related to monocyte infiltration and that isolated T-cell infiltration has less acute functional impact. This relationship may be useful in assigning acute clinical relevance to biopsy findings.

Colorectal cancer imaging with iodine-123-labeled CEA monoclonal antibody fragments.

This prospective, randomized multicenter study in 62 patients was designed to evaluate the efficacy and safety of radioimmunodetection (RAID) with 123I-labeled fragments, F(ab')2 and Fab', of IMMU-4, an anti-CEA monoclonal antibody (Immu-RAID-CEA). It was found that ImmuRAID-CEA was safe and disclosed colorectal cancer sites at least 1 cm in size. The positive predictive value by lesions was 77% initially, and increased to 91% after 7 mo of follow-up. Only one patient developed a low level of HAMA. In 17 patients with 32 surgically confirmed lesions, there were 9% true-positive lesions for CT when RAID was false-negative, and 22% for RAID when CT was false-negative. Either CT or RAID detected all 32 lesions. In this small series, therefore, RAID was shown to complement CT findings by confirming suspected tumors and disclosing new lesions which had previously been occult.

Thoracic extramedullary hematopoiesis in sickle-cell disease.

The radiographic manifestations of thoracic extramedullary hematopoiesis are unilateral or bilateral, smooth, sharply delineated, often lobulated paraspinal masses without erosion of the vertebral bodies or ribs, sometimes associated with subpleural, paracostal masses. These radiographic findings were encountered in eight adult patients with homozygous sickle-cell disease. In one patient, the masses encompassed essentially the entire thoracic paravertebral area bilaterally. In the other seven patients, the masses were unilateral and limited to the region of the eighth to the 12th thoracic vertebrae. Two of the eight patients had lateral subpleural masses that were not contiguous with the paraspinal masses and that were located medial to the lateral portions of the ribs. Follow-up in seven of the cases ranged from 2 to 15 years and demonstrated, after relatively rapid growth initially, either no change in size or slow growth. Each patient was asymptomatic with regard to the thoracic masses. Histologic verification was not available in any case because of the lack of clinical indication for invasive diagnostic or therapeutic procedures. The presence of well-defined unilateral or bilateral paraspinal masses and/or paracostal masses in patients with homozygous sickle-cell disease and without related symptoms should alert one to the possibility of the presence of extramedullary hematopoiesis. These masses tend to be slow-growing and should not be subjected to aggressive diagnostic and therapeutic measures.

Determination of sequestrum activity by SPECT with CT correlation in chronic osteomyelitis of the head and neck.

Use of single photon emission computerized tomography (SPECT) with Tc 99m HDP for localization of an active sequestrum in a case of chronic head and neck osteomyelitis is described. Correlation with x-ray computed tomography (CT) for anatomy was helpful for surgical debridement. The potential of the complementary roles of SPECT and CT in chronic head and neck osteomyelitis for assessing sequestrum activity and anatomy prior to surgery is discussed.

Acquired functional asplenia in sarcoidosis.

Sarcoidosis is a recently identified cause of functional asplenia that can be diagnosed by radionuclide imaging. A 31-year-old woman with a five-year history of histologically compatible sarcoidosis was found to have nonvisualization of the spleen on technetium 99m sulfur colloid (radiopharmaceutical) liver-spleen scan. This scintigraphic finding was accompanied by poikilocytosis and Howell-Jolly bodies in the peripheral blood smear. A subsequent gallium 67 citrate scan reflected an abnormal increase in concentration of activity in the spleen, suggesting an active inflammatory process.Based upon this constellation of findings, it was concluded that acquired functional asplenia is the result of reticuloendothelial cell replacement via infiltration of the spleen by epithelioid cell granulomas of active sarcoidosis. This case also illustrates the reversibility of functional asplenia of sarcoidosis following adrenocorticosteroid therapy. Functional asplenia in sarcoidosis is now found to have a recognizable radionuclide imaging pattern.