Clinical use of progesterone in human sperm preparation media for increasing IVF success.

RESEARCH QUESTION: Can the addition of progesterone and neurotensin, molecular agents found in the female reproductive tract, after sperm washing increase the fertilization potential of human spermatozoa? DESIGN: (i) Cohort study of 24 men. Spermatozoa selected by swim-up were incubated in either progesterone or neurotensin (0.1-100 µM) for 1-4 h, and hyperactive motility and binding to hyaluronan (0.1-100 µM) were assessed. The effect of progesterone 10 µM on sperm function was assessed in a blinded manner, including: hyperactive motility, binding to hyaluronan, tyrosine phosphorylation, acrosome reaction and oxidative DNA damage. (i) Embryo safety testing [one-cell mouse embryo assay (MEA), endotoxin and sterility counts (n = 3)] in preclinical embryo models of IVF (murine and porcine, n = 7 each model) and a small preliminary human study (n = 4) of couples undergoing standard IVF with oocytes inseminated with spermatozoa ± 10 µM progesterone. RESULTS: Progesterone 10 µM increased sperm binding to hyaluronan, hyperactive motility and tyrosine phosphorylation (all P < 0.05). Neurotensin had no effect (P > 0.05). Progesterone 10 µM in human embryo culture media passed embryo safety testing (MEA, endotoxin concentration and sterility plate count). In preclinical models of IVF, the exposure of spermatozoa to progesterone 10 µM and oocytes to progesterone 1 µM was not detrimental, and increased the fertilization rate in mice and the blastocyst cell number in mice and pigs (all P ≤ 0.03). In humans, every transferred blastocyst that had been produced from spermatozoa exposed to progesterone resulted in a live birth. CONCLUSION: The addition of progesterone to sperm preparation media shows promise as an adjunct to current methods for increasing fertilization potential. Randomized controlled trials are required to determine the clinical utility of progesterone for improving IVF outcomes.

PIEZO-ICSI increases fertilization rates compared with standard ICSI: a prospective cohort study.

RESEARCH QUESTION: Is PIEZO-intracytoplasmic sperm injection (ICSI) coupled with a new novel operational fluid (perfluoro-n-octane) superior to standard ICSI? DESIGN: A cohort of patients (n = 69) undertaking microinjection were recruited between January and November 2019 and were then prospectively case-matched. Patients required six or more mature oocytes for inclusion in the study. PIEZO-ICSI uses high-speed microinjection drilling to penetrate the zona and oolemma and deposit the spermatozoa into the cytoplasm, compared with the traditional 'cutting' action of ICSI. The primary outcome was fertilization, with secondary outcomes including oocyte degeneration, abnormal fertilization, embryo cryopreservation and embryo utilization. RESULTS: PIEZO-ICSI resulted in significantly higher fertilization rates (80.5 ± 2.4% vs 65.8 ± 2.3%, P < 0.0001) and lower oocyte degeneration rates (4.4 ± 1.3% vs 8.6 ± 1.2%, P = 0.019) and abnormal fertilization rates (2.9 ± 1.1% vs 7.4 ± 1.1%; P = 0.003) compared with standard ICSI. This improvement in fertilization was of most benefit in patients aged ≥38 years. This increase in fertilization increased the number of good quality embryos that were available for cryopreservation/transfer (3.8 ± 0.2 vs 3.1 ± 0.2; P = 0.038), such that patients on average had one extra usable embryo per cycle compared with standard ICSI. There were no differences to Day 5 embryo development or clinical pregnancy from fresh embryo transfer (57.1% PIEZO-ICSI vs 60.0% ICSI) between microinjection methods, although pregnancy outcomes were underpowered. CONCLUSIONS: PIEZO-ICSI significantly increased fertilization rates, thereby increasing the number of embryos available for cryopreservation compared with standard ICSI. Further prospective studies assessing cumulative pregnancy rates are warranted.

Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors.

Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreER RosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-ß) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases.

Mini-sternotomy vs right anterior thoracotomy for aortic valve replacement.

BACKGROUND: While minimally invasive techniques for aortic valve replacement (AVR) have been shown to be safe, limited data exist comparing the varying approaches. This study aimed to compare the outcomes between two minimally invasive approaches for AVR: mini-sternotomy (MS) and right anterior thoracotomy (RAT). MATERIALS AND METHODS: A systematic search of MEDLINE, EMBASE, and OVID was conducted for the period 1990-2019. Nine observational studies (n = 2926 patients) met the inclusion criteria. RESULTS: There was no difference in operative mortality between MS and RAT (odds ratio [OR]: 0.87, 95% confidence interval [CI]: 0.41-1.85; P = .709). Meta-analyses favored MS over RAT in reoperation for bleeding (OR: 0.42, 95% CI: 0.28-0.63; P < .001), aortic cross-clamp time (standardized mean difference [SMD]: -0.12, 95% CI: -0.20 to 0.029; P = .009), and the rate of conversion to sternotomy (OR: 0.32, 95% CI: 0.11-0.93; P = .036). The rate of permanent pacemaker insertion approached borderline significance in favor of MS (OR: 0.54, 95% CI: 0.26-1.12; P = .097). In-hospital outcomes of stroke, atrial fibrillation, and surgical site infection were similar between the two groups. The length of hospital stay was shorter for RAT (SMD: 0.12, 95% CI: 0.027-0.22; P = .012) and the length of postoperative ventilation was borderline significant in favor of RAT (SMD: 0.16, 95% CI: -0.027 to 0.34; P = .095). CONCLUSIONS: This study highlights important differences in short-term outcomes between MS and RAT as approaches for AVR. This has important implications for patient selection, especially in the elderly, where such approaches are becoming more common-place.

Transthoracic clamp versus endoaortic balloon occlusion in minimally invasive mitral valve surgery: a systematic review and meta-analysis.

This systematic review and meta-analysis aims to determine outcomes following aortic occlusion with the transthoracic clamp (TTC) versus endoaortic balloon occlusion (EABO) in patients undergoing minimally invasive mitral valve surgery. A subgroup analysis compares TTC to EABO with femoral cannulation separately from EABO with aortic cannulation. We searched Medline and Embase up to December 2018. Two people independently and in duplicate screened title and abstracts, full-text reports, extracted data and assessed the risk of bias using the Cochrane risk-of-bias tool for non-randomized studies. We identified 1564 reports from which 11 observational studies with 4181 participants met the inclusion criteria. We found no evidence of difference in the risk of postoperative death or cerebrovascular accident (CVA) between the 2 techniques. Evidence for a reduction in aortic dissection with TTC was found: 4 of 1590 for the TTC group vs 19 of 2492 for the EABO group [risk ratio 0.33, 95% confidence interval (CI) 0.12-0.93; P = 0.04]. There was no difference in aortic cross-clamp (AoX) time between TTC and EABO [mean difference (MD) -5.17 min, 95% CI -12.40 to 2.06; P = 0.16]. TTC was associated with a shorter AoX time compared to EABO with femoral cannulation (MD -9.26 min, 95% CI -17.00 to -1.52; P = 0.02). EABO with aortic cannulation was associated with a shorter AoX time compared to TTC (MD 7.77 min, 95% CI 3.29-12.26; P < 0.001). There was no difference in cardiopulmonary bypass (CPB) time between TTC and EABO with aortic cannulation (MD -4.98 min, 95% CI -14.41 to 4.45; P = 0.3). TTC was associated with a shorter CPB time compared to EABO with femoral cannulation (MD -10.08 min, 95% CI -19.93 to -0.22; P = 0.05). Despite a higher risk of aortic dissection with EABO, the rates of survival and cerebrovascular accident across the 2 techniques are similar in minimally invasive mitral valve surgery.

Sequential clomiphene/corifollitrophin alpha as a technique for mild controlled ovarian hyperstimulation in IVF: a proof of concept study.

PURPOSE: Mild controlled ovarian hyperstimulation (COH), combined with oocyte retrieval (OR) under local anaesthesia (LA), may provide low-impact IVF. Since a single injection of corifollitrophin alfa (CFA) provides 7 days of COH, we hypothesised that clomiphene-citrate (CC) followed by CFA may provide adequate COH response from one single FSH injection. Therefore, the aim was to assess IVF outcomes after a novel clomiphene citrate/CFA (CC/CFA) protocol, compared to women undergoing standard rFSH COH protocols (good prognosis comparative cohort:GPCC) in a 1:2 matched design. MATERIALS AND METHODS: In this pilot study of 25 patients (ANZCTR id:ACTRN12612000740897, MINIVA:Minimal_Stimulation_in_IVF), we examined the effectiveness of oral clomiphene (100 mg-days 2-6) followed by CFA in a GnRH antagonist protocol producing a single injection COH stimulation regime. All OR were conducted under LA pre-ovarian block. Cycle outcomes were compared to a matched good prognosis comparative cohort (GPCC) undergoing standard rFSH COH. RESULTS: Mild stimulation was achieved with less oocytes being collected compared to the GPCC (6.4 ± 0.7 vs. 10.7 ± 0.9, p < 0.001), resulting in a reduced number of good quality embryos available for transfer/cryopreservation (3.7 ± 0.6 vs. 5.7 ± 0.5, p = 0.01). While embryo quality was similar between the two groups, endometrial thickness was significantly lower in the group receiving CC/CFA. Pregnancy rates were significantly lower in the CC/CFA cohort compared to GPCC (31.8 vs. 57.1%, p = 0.04) and 44% of CC/CFA participants required supplemental rFSH in order to achieve the hCG trigger criteria. CONCLUSION: Sequential clomiphene CFA protocol does not appear to be an optimal regime for low impact IVF treatment as it does not provide adequate COH from a single CFA injection and results in lower fresh embryo transfer pregnancy rates and fewer embryos for cryopreservation.

Children and young people with diabetes: recognition and management.

Diabetes mellitus (type 1) is an autoimmune condition leading to absolute insulin deficiency resulting in hyperglycaemia and its associated manifestations. It is the most common type of diabetes seen in children, characterised by the 4Ts (toilet, thinner, thirsty, tired). The signs and symptoms can sometimes be non-specific and a delay or missed diagnosis may be catastrophic to the health of the child. Children with an established diagnosis of diabetes often present to the health service with issues such as hypoglycaemia, hyperglycaemia, or diabetic ketoacidosis. The condition requires life-long monitoring and strict control of blood glucose levels with insulin replacement therapy, with the aim of achieving an HbA1c level of 48 mmol/mol. There are often physical and psychosocial issues that arise from the diagnosis leading to poor control. Nurses working in different clinical settings play a vital role in raising suspicions of diabetes leading to timely diagnosis and rapid initiation of treatment. They are best placed to provide essential support in helping children and their families to come to terms with the diagnosis, as well as manage this chronic condition by addressing the common issues that arise in the ongoing management, with a particular emphasis on managing the various day-to-day challenges. Two case studies are included to highlight some of the challenges that nurses may encounter while managing children with diabetes.

Results of complex aortic stent grafting of abdominal aortic aneurysms stratified according to the proximal landing zone using the Society for Vascular Surgery classification.

BACKGROUND: Advances in endovascular technology have led to the successful treatment of complex abdominal aortic aneurysms. However, there is currently no consensus on what constitutes a juxtarenal, pararenal, or suprarenal aneurysm. There is emerging evidence that the extent of the aneurysm repair is associated with outcome. We compare the outcomes of 150 consecutive patients treated with a fenestrated or branched stent graft and present the data stratified according to the Society for Vascular Surgery classification based on proximal anatomic landing zones. METHODS: A prospectively collected database of consecutive patients undergoing fenestrated or branched stent graft insertion in a tertiary center between 2008 and 2013 was retrospectively analyzed. Aneurysms were subdivided into zones according to where the area of proximal seal could be achieved in relation to the visceral arteries. Zone 8 covers the renal arteries, zone 7 covers the superior mesenteric artery, and zone 6 covers the celiac axis. Patient demographics, operative variables, mortality, and major morbidity were analyzed by univariate and multivariate analysis to assess for differences between zones. RESULTS: During the study period, 150 patients were treated. There were 49 in zone 8, 76 in zone 7, and 25 in zone 6. Prior aortic surgery had been performed in 19 patients, which included 11 patients with previous endovascular aneurysm repairs. There was significantly increased blood loss (P < .001), operative time (P < .0001), total hospital stay (P = .018), and intensive care unit stay (P < .0001) as the zones ascended the aorta. There were 14 inpatient deaths recorded across all zones with a 30-day mortality rate of 8%. Logistic regression analysis for 30 day mortality showed a significant increase as the zones ascended (P = .007). Kaplan-Meier analysis showed that 5-year survival significantly deteriorated as the zones ascended (P = .039), with no significant difference in the freedom from reintervention curves between zones (P = .37). CONCLUSIONS: We have shown that the extent of the aneurysm repair as determined by the proximal sealing zone is associated with outcome. Mortality, operative duration, blood loss, and hospital stay all significantly increased as the zones ascended. These data also validate the use of the proposed new classification based on aortic anatomy.

Temporary Perfusion Branches to Decrease Spinal Cord Ischemia in the Endovascular Treatment of Thoraco-Abdominal Aortic Aneurysms: Based on a Presentation at the 2013 VEITH Symposium, November 19-23, 2013 (New York, NY, USA).

BACKGROUND: Spinal cord ischemia (SCI) is one of the most feared complications following the repair of thoraco- abdominal aortic aneurysms (TAAA). Endovascular repair of TAAA is now possible with branched stent grafts, but spinal cord ischaemia rates are still unacceptably high. A number of techniques have been utilized to reduce these levels, however, SCI remains a challenge to endovascular repair of TAAA. The use of sac perfusion branches aims to reduce the incidence of this catastrophic complication. METHODS: A retrospective analysis of all patients undergoing branched endovascular aortic repair for all thoraco-abdominal aneurysms (TAAA) using custom made devices during January 2008 to August 2014. We describe a two staged technique in which perfusion of segmental vessels is maintained by a temporary endoleak through an open perfusion branch, incorporated within the branched stent graft, followed by a closure of this branch at a later date to complete exclusion of the aneurysm. RESULTS: Forty-seven patients underwent TAAA repair. Twenty-five (53%) had a two-stage procedure using either a sac perfusion branch or a target vessel to perfuse the sac. Nine patients (19.15%) suffered some form of SCI with eight patients having temporary SCI (lasting less than 72 hours) and one patient having permanent SCI. Of eight patients that had temporary spinal cord ischemia, all had a perfusion strategy. There was one case of permanent SCI (2.13%). CONCLUSION: Sac perfusion branches provide a safe method for preventing SCI, however this needs to be used in conjunction with controlling MAP and CSF drainage.

Successful stent graft repair of multiple thoracoabdominal mycotic aortic aneurysms in the presence of Kommerell diverticulum and complicated by aortoesophageal fistula.

We describe the management of a woman who presented with synchronous mycotic aortic aneurysms of the aortic arch in the presence of Kommerell diverticulum, the distal thoracic, and the juxtarenal aorta. A staged stent graft repair was undertaken due to rapid expansion of the aneurysms, which involved placement of multiple thoracic quadruple-fenestrated and infrarenal bifurcated stent grafts. Despite complications of an aortoesophageal fistula and transitory spinal cord ischemia, she has been managed successfully and is doing well at 36 months. This case illustrates that stent graft repair of mycotic aneurysms can offer a successful treatment option in selected patients.

A rare and potentially fatal cause of popliteal fossa swelling.

Popliteal vein aneurysms (PVAs) represent a rare form of venous aneurysm and necessitate prompt diagnosis and management due to their well-established role as a source of pulmonary emboli. Surgical repair is usually curative; we present the case of a 47-year-old woman with a recurrent PVA presenting as a soft popliteal fossa mass with associated sensory deficit affecting the right foot, 4 years after initial operative repair. Venous duplex imaging demonstrated a saccular aneurysm originating from the posterior wall of the right popliteal vein. In light of the long-term risk of potentially life-threatening pulmonary embolism, surgical repair was undertaken by tangential aneurysmectomy and long saphenous vein patch venoplasty. This case emphasises the importance of including PVA in the differential diagnosis of popliteal fossa swellings as well as the need for long-term follow-up to detect late recurrence with subsequent avoidance of the otherwise significant potential morbidity and mortality.

Slow freezing and vitrification of mouse morula and early blastocysts.

PURPOSE: To assess the relative success of morula and early blastocyst slow freezing and vitrification in regards to survival and implantation rates utilising protocols which could be clinically implemented as a viable alternative to expanded blastocyst stage freezing. METHODS: Mouse morula and early blastocysts were either slow frozen/thawed or vitrified/warmed. Their subsequent survival, blastocyst development and blastocyst cell number and allocation to either the inner cell mass, trophectoderm or epiblast was assessed. In addition blastocysts were also transferred to pseudopregnant recipients and implantation and fetal development was determined. RESULTS: Vitrification of both morula and early blastocysts resulted in significantly higher rates of survival and blastocyst development compared to slow freezing. In addition slow frozen early blastocysts had significantly reduced blastocyst cell number compared to control however vitrified morula and early blasocyts and slow frozen morula had equivocal blastocyst cell numbers. Transfer of blastocysts from both methods of cryopreservation resulted in similar implantation rates however the placentas created from slow frozen early blastocysts were significantly lighter than control (95.5 g ± 5.4 vs. 122.0 g ± 4.2 respectively). CONCLUSIONS: Vitrification resulted in significantly higher rates of morula and early blastocyst survival and blastocyst development compared to slow freezing. In addition this study has validated the use of a closed DMSO free vitrification protocol which could then be investigated for use in the clinical setting as an alternative to expanded blastocyst freezing.

Does obesity really matter? The impact of BMI on embryo quality and pregnancy outcomes after IVF in women aged ≤38 years.

BACKGROUND: The increasing prevalence of obesity in women of child-bearing age is of growing concern in the health community. Obesity is associated with sub-optimal reproductive performance; therefore, it is understandable that the number of young women with elevated body mass index (BMI) accessing assisted reproductive treatment (ART) is on the rise. Consequently, this study not only assessed the impact of BMI on fertilisation rates, embryo development and freezing during ART in women aged ≤38 years but also determined their subsequent pregnancy and delivery rates. METHODS: Data were retrospectively analysed from all cycles initiated in 2006/2007 for women aged ≤38 years. The BMI categorisations were as follows: normal - 18.5-24.9 kg/m(2) ; overweight - 25-29.9 kg/m(2) ; obese - 30-34.9 kg/m(2) ; morbidly obese class I - 35-39.9 kg/m(2) ; morbidly obese class П -≥40 kg/m(2) . RESULTS: Obese and morbidly obese women required a significantly higher follicle stimulating hormone start dose than normal BMI women; however, they obtained significantly fewer oocytes (P < 0.05). Although BMI did not affect embryo development, morbidly obese class Π women had significantly reduced pregnancy rates compared to normal BMI women (30.5 vs 41.7%, respectively; P < 0.05). Furthermore, increasing BMI was positively correlated to increasing rates of preterm delivery (P < 0.05). Increasing BMI was also positively correlated to increasing delivery rates of singleton term macrosomic offspring (≥4000 g). CONCLUSION: Obesity in women aged≤38 years does not affect embryo development; however, it does reduce clinical pregnancy rates in women with a BMI≥40 and increases rates of preterm labour and delivery of macrosomic offspring.

Appendicitis in an incisional hernia after radical nephrectomy: a case report.

Appendicitis is one of the most common surgical emergencies, and is usually diagnosed clinically on point tenderness in the right iliac fossa. The diagnosis can be difficult if there is abnormal anatomy. This case looks into the presentation of appendicitis within an incisional hernia secondary to radical nephrectomy. Appendicitis may present within hernias, and there should be a low threshold for computed tomography assessment of hernias when there is clinical doubt about the symptoms associated with the hernia.

A comparison of two in vitro maturation media for use with adult porcine oocytes for adult somatic cell nuclear transfer.

Two media used to mature adult porcine oocytes for somatic cell nuclear transfer were compared. In the first experiment, parthenogenetic embryos were produced using a maturation medium used by us previously to clone pigs (OMM199) and that described by Kühholzer et al. (2001) to transport oocytes overnight (BOMED). There was no difference in maturation rates between the two different media. However, BOMED medium increased the percentage of parthenogenetic embryos that developed to the blastocyst stage compared with OMM199 (49% vs. 29%, respectively). In a second experiment, BOMED medium increased the percentage of SCNT embryos that developed to the blastocyst stage compared with OMM199 (22% vs. 8%, respectively). The efficiency of our cloning protocol using adult oocytes matured in BOMED medium was then determined by transferring SCNT embryos reconstructed using adult fibroblasts to synchronized recipients. Primary cultures of adult fibroblasts were obtained from two adult male pigs and used for SCNT (passages 2-4). Between 82 and 146 fused couplets were transferred to seven recipients synchronized 1 day behind the embryos. Five recipients (71% pregnancy rate) subsequently farrowed a total of 23 piglets (4.4 average litter size). Overall efficiencies (liveborn/embryos transferred) were 3.2% for all transfers and 4.3% for animals that gave birth.

Predominance of Th2 response in human abdominal aortic aneurysm: mistaken identity for IL-4-producing NK and NKT cells?

Abdominal aortic aneurysm (AAA) is a complex remodeling process that involves both synthesis and degradation of extracellular matrix proteins in the aortic wall, leading to decreased tensile strength, progressive dilation and eventual rupture. Chronic inflammation, increased local production of elastin-degrading proteases by inflammatory cells and destruction of medial elastic lamellae play important roles in aneurysm progression. Neovascularization in all layers of the arterial wall is prominent and angiogenesis can facilitate chronic inflammation. It is still unclear what initiates aneurysmal dilation and what determines its progression. The complex nature of the process has defied elucidation. Apart from macrophages, the predominant immune cell infiltrates reported so far are CD3(+)T cells that express CD4 and CD8. Infiltrates of type 2 Th cells and their production of IL-4 and IL-5 have been implicated in AAA development. However, NKT and NK cells have a Th0 cytokine profile and can also produce type 2 as well as type 1 (IL-2 and IFNgamma) cytokines. We have demonstrated the presence of NK and NKT cells in AAA tissue. With their growing importance in autoimmunity and transplantation, they may play a role in AAA development. Therefore, there is a need to use a combination of T and NK markers to fully characterize both innate and adaptive lymphoid cell subsets in local inflammatory infiltrates in order to elucidate their roles in AAA progression.

Atherosclerotic abdominal aortic aneurysm and the interaction between autologous human plaque-derived vascular smooth muscle cells, type 1 NKT, and helper T cells.

Immune cell infiltration, vascular smooth muscle cell (VSMC) proliferation, and apoptosis are pathological hallmarks of atherosclerosis. The multifocal, chronic, and inflammatory nature of this disease of the cardiovascular system complicates targeted cellular therapy and emphasizes the need to understand the role and interaction of immune cells with VSMCs. We characterized the immune cell subsets present in human atherosclerotic tissue derived from atherosclerotic abdominal aortic aneurysm (AAA) and expanded them to study their interaction with autologous plaque-derived VSMCs in vitro. We show here that apart from T lymphocytes, plaque infiltrates consist of lots of NK cells and significant proportions of NKT cells that express T cell receptor (TCR) alphabeta, CD4, and the NK markers CD56 and CD161. The infiltrates are predominantly IFN-gamma-producing Type 1 lymphoid cells. When cocultured, the T and NKT cells adhere to VSMCs. CD4+ T cells enhance VSMC proliferation. VSMCs in turn enhance CD4+CD161+ NKT but not CD4+ or CD8+ T cell proliferation. CD4+CD161+ NKT cells inhibit VSMC proliferation by inducing apoptosis. Our results suggest that the interactions of Type 1 CD4+ T and CD4+CD161+ NKT cells with VSMCs may regulate VSMC proliferation and death respectively in atherosclerosis and the balance of these interactions could determine plaque stability.

Ovine ooplasm directs initial nucleolar assembly in embryos cloned from ovine, bovine, and porcine cells.

Here we present ultrastructural and immunocytochemical evidence that ovine ooplasm is directing the initial assembly of the nucleolus independent of the species of the nuclear donor. Intergeneric porcine-ovine somatic cell nuclear transfer (SCNT) and intrageneric ovine-ovine SCNT embryos were constructed and the nucleolus ultrastructure and nucleolus associated rRNA synthesis examined in 1-, 2-, 4-, early 8-, late 8-, and 16-cell embryos using transmission electron microscopy (TEM) and light microscopical autoradiography. In addition, immunocytochemical localization by confocal microscopy of nucleolin, a key protein involved in processing rRNA transcripts, was performed on early 8-, late 8-, and 16-cell embryos for both groups of SCNT embryos. Intergeneric porcine-ovine SCNT embryos exhibited nucleolar precursor bodies (NPBs) of an ovine (ruminant) ultrastructure, but no active rRNA producing fibrillo-granular nucleoli at any of the stages. Unusually, cytoplasmic organelles were located inside the nucleus of two porcine-ovine SCNT embryos. The ovine-ovine SCNT embryos, on the other hand, revealed fibrillo-granular nucleoli in 16-cell embryos. In parallel, autoradiographic labeling over the nucleoplasm, and in particular, the nulcleoli was detected. Bovine-ovine SCNT embryos at the eight-cell stage were examined for nucleolar morphology and exhibited ruminant-type NPBs as well as structures that appeared as fibrillar material surrounded by a rim of electron dense granules, perhaps formerly of nucleolar origin. Nucleolin was localized throughout the nucleoplasm and with particular intensity around the presumptive nucleolar compartments for all developmental stages examined in porcine-ovine and ovine-ovine SCNT embryos. In conclusion, this study suggests that factors within the ovine ooplasm are playing a role in the initial assembly of the embryonic nucleolus in intrageneric SCNT embryos.

NKT cell subsets in infection and inflammation.

We recently identified two stably expressed cell surface markers, IL-18R and ST2L, which are selectively expressed on T1/NK1 and T2/NK2 cells, respectively. Here we use these molecules in direct ex vivo analysis of PBMCs from patients with AIDS, psoriasis (PS) atherosclerosis and to show the importance of these markers as determinants of the functional dichotomy of lymphocyte subsets, in particular NKT. In a cohort of 22 HIV patients made up of a mixture of long term non-progressors, seroconvertors, progressors and asymptomatics, we found a clear NKT1 to NKT2 shift (P=0.001) in the HIV-infected individuals. We also show a predominance of NKT2 cells over NKT1 cells in the PBMCs of patients with mild to moderate PS (N=13, P=0.005) but not in atopic dermatitis or healthy controls. However, in patients (N=6) requiring surgery for aneurysm, a predominance of Type 1 (IL-18R(+)) NKT lymphocytes over NKT2 was detected among infiltrating lymphocytes isolated from atherosclerotic plaques. Our data therefore demonstrate that ST2L and IL-18R could serve as important determinants of the immune status of human diseases.