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OBJECTIVE: The purpose of this study was to test whether treating periodontal disease (PD) in pregnancy will reduce the incidence of spontaneous preterm delivery (SPTD) at < or = 35 weeks of gestation. STUDY DESIGN: A multicenter, randomized clinical trial was performed. Subjects with PD were randomized to scaling and root planing (active) or tooth polishing (control). The primary outcome was the occurrence of SPTD at <35 weeks of gestation. RESULTS: We screened 3563 subjects for PD; the prevalence of PD was 50%. Seven hundred fifty-seven subjects were assigned randomly; 378 subjects were assigned to the active group, and 379 subjects were assigned to the placebo group. Active treatment did not reduce the risk of SPTD at <35 weeks of gestation (relative risk, 1.19; 95% confidence interval [CI], 0.62-2.28) or composite neonatal morbidity (relative risk, 1.30; 95% CI, 0.83-2.04). There was a suggestion of an increase in the risk of indicated SPTD at <35 weeks of gestation in those subjects who received active treatment (relative risk, 3.01; 95% CI, 0.95-4.24). CONCLUSION: Treating periodontal disease does not reduce the incidence of SPTD. [ABSTRACT FROM AUTHOR]

Cyclical menstrual psychosis is an uncommon, generally a self-limiting mental illness that occurs only in females. It is associated with other menstruation-related disorders and stressful psychogenic factors. Nonetheless, many cases remain unrecognized due to poor awareness of its presence. A young female who presented with psychotic and mood symptoms during each cycle of menstruation was admitted to the psychiatric inpatient unit. There was severe disruption in her activities of daily living and socio-occupational functioning. Treatment involved bio-psycho-social approach in collaboration with Ob-Gyn team with symptoms responding well to a combination of valproic acid and risperidone. Severe affective instability with evident psychosis during menstrual cycle should be evaluated for cyclical menstrual psychosis., Competing Interests: Conflicts of Interest None

The purpose of this study was to evaluate the outcomes of the Boston type 1 keratoprosthesis (Kpro-1) in eyes with failed keratoplasty. A retrospective review was performed of every patient treated with a Kpro-1 at a tertiary eye care center between January 1, 2008 and July 1, 2013. Eyes with a failed keratoplasty originally performed for corneal edema, trauma, or keratoconus were included in the statistical analysis. The main outcome measures were visual outcome, prosthesis retention, and postoperative complications. Twenty-four eyes met the inclusion criteria, including 13 eyes with corneal edema, 8 eyes with trauma, and 3 eyes with keratoconus. After a mean follow-up period of 28.9 months (range 7-63 months), the median best corrected visual acuity (BCVA) was 20/125. The BCVA was ≥ 20/40 in 4 (16.7 %) eyes, ≥ 20/70 in 9 (37.5 %) eyes, and ≥ 20/200 in 14 (58.3 %) eyes. Overall, the postoperative BCVA improved in 17 (70.9 %) eyes, was unchanged in 3 (12.5 %) eyes, and was worse in 4 (16.7 %) eyes. The initial Kpro-1 was retained in 22 (91.7 %) eyes, and was successfully repeated in the other 2 eyes. One or more serious prosthesis- or sight-threatening complications occurred in 8 (33.3 %) eyes. These included 1 case of wound dehiscence leading to prosthesis extrusion, 1 case of fungal keratitis leading to prosthesis extrusion, 4 cases of endophthalmitis, and 5 retinal detachments. The Boston Kpro-1 is associated with an excellent prognosis for prosthesis retention and satisfactory visual improvement in eyes with previous failed keratoplasty.

Objective: To establish current knowledge of patient safety and quality of care in developing countries in Southeast Asia, current interventions and the knowledge gaps., Study Design: Systematic review and narrative synthesis., Data Sources: Key words, synonyms and subject headings were used to search seven electronic databases in addition to manual searching of relevant journals., Data Synthesis: Titles and abstracts of publications between 1990 and 2014 were screened by two reviewers and checked by a third. Full text articles were screened against the eligibility criteria. Data on design, methods and key findings were extracted and synthesized., Results: Four inter-related safety and quality concerns were evident from 33 publications: (i) the risk of patient infection in healthcare delivery, (ii) medications errors/use, (iii) the quality and provision of maternal and perinatal care and (iv) the quality of healthcare provision overall., Conclusions: Large-scale prevalence studies are needed to identify the full range of safety and quality problems in developing countries in Southeast Asia. Sharing lessons learnt from extensive quality and safety work conducted in industrialized nations may contribute to significant improvements. Yet the applicability of interventions utilized in developed countries to the political and social context in this region must be considered. Strategies to facilitate the collection of robust safety and quality data in the context of limited resources and the local context in each country are needed., (© The Author 2015. Published by Oxford University Press in association with the International Society for Quality in Health Care; all rights reserved.)

Purpose: To evaluate the outcome of the Boston type 1 keratoprosthesis (Kpro-1) in eyes with failed interventions for chemical and thermal injury., Methods: A retrospective review was performed of every eye with chemical or thermal injury that was treated with a Kpro-1 at a tertiary eye care center between January 1, 2008 and July 1, 2013. The main outcome measures were visual outcome, prosthesis retention, and postoperative complications., Results: Nine eyes met the inclusion criteria, including 7 eyes with alkali burns, 1 eye with an acid burn, and 1 eye with a thermal burn. After a mean follow-up of 40.7 months (range, 29-60 months), the median best-corrected visual acuity was 20/60 (range, 20/15 to no light perception). One eye was ≥20/20, 3 eyes were ≥20/40, and 6 eyes were ≥20/70. The initial Kpro-1 prosthesis was retained in 7 (77.7%) eyes and successfully replaced in the other 2 eyes. One or more serious complications occurred in 6 (66.7%) eyes. These included 2 cases of sterile corneal ulceration with prosthesis extrusion, 2 cases of microbial keratitis (1 bacterial and 1 fungal), 2 cases of bacterial endophthalmitis, and 2 cases of retinal detachments. These complications contributed to visual outcomes of hand motions in 2 eyes and no light perception in 1 eye., Conclusions: The Boston Kpro-1 is associated with highly satisfactory visual outcomes and prosthesis retention in most cases of severe chemical or thermal injury. Serious complications are common and may compromise the final outcome.

Purpose: Caveolin-1 (Cav-1), the signature protein of caveolae, modulates inflammatory responses, and innate immunity. However, Cav-1's role in retinal inflammation has not been rigorously tested. In this study, we examined the effect of Cav-1 ablation on the sensitivity of the retina to inflammation., Methods: Cav-1 knockout (KO) mice were challenged by intravitreal injection of lipopolysaccharide (LPS) and inflammatory cell recruitment was assessed by flow cytometry and immunohistochemistry. Leukostasis was assessed in retinal flatmounts after perfusion with FITC-labeled Concanavalin A (FITC-ConA). Chemoattractants were measured by multiplex immunoassays. Blood-retinal barrier (BRB) breakdown was assessed quantitatively by a FITC-dextran permeability assay. The ratio of extravascular to total immune cells was determined by CD45 immunohistochemistry of retinal flatmounts., Results: Inflammatory challenge resulted in significant blunting of proinflammatory cytokine (monocyte chemoattractant protein-1 [MCP-1/CCL2], CXCL1/KC, IL-6, and IL-1β) responses as well as reduced inflammatory BRB breakdown in Cav-1 KO retinas. Paradoxically, Cav-1 deficiency resulted in significantly increased recruitment of immune cells compared with controls as well as increased leukostasis. A similar ratio of extravascular/total leukocytes were found in Cav-1 KO and wild-type (WT) retinas suggesting that Cav-1 deficient leukocytes were as competent to extravasate as those from WT mice. We found increased levels of circulating immune cells in naïve (not challenged with LPS) Cav-1 KO mice compared with controls., Conclusions: Caveolin-1 paradoxically modulates inflammatory signaling and leukocyte infiltration through distinct mechanisms. We hypothesize that Cav-1 expression may enhance inflammatory signaling while at the same time supporting the physical properties of the BRB., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Objective: To report a case of acute abdomen secondary to torsion of uterine remnant leiomyoma and ipsilateral adnexa in a woman with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome and known history of bilateral uterine remnant leiomyomas., Design: Case report., Setting: Tertiary care center., Patient(s): A 40-year-old nulligravid woman with MRKH syndrome and a known history of bilateral uterine remnant leiomyomas with a surgical abdomen., Surgery: right salpingo-oopherectomy, excision of right and left hemiuteri with pedunculated leiomyomas, and left salpingectomy., Main Outcome Measure(s): Definitive therapy with preservation of premenopausal state., Result(s): First known case of acute surgical presentation secondary to torsion of uterine remnant leiomyoma and ipsilateral adnexa in a woman with MRKH syndrome and bilateral voluminous uterine remnant leiomyomas without any plan for elective surgical intervention., Conclusion(s): Elective surgical removal of uterine remnant leiomyoma for women with MRKH syndrome can prevent the complication of torsion as well as prevent ovarian resection in premenopausal women. Torsion should be considered in the differential diagnosis in a woman with MRKH syndrome and known history of leiomyomas who presents with acute abdomen., (Published by Elsevier Inc.)

Purpose: The aim of this study was to evaluate and compare the outcomes of Boston keratoprosthesis type 1 (Kpro-1) in eyes with herpes simplex virus (HSV) and herpes zoster virus (HZV) keratopathy., Methods: A retrospective review was performed of the medical records of every patient treated with a Boston Kpro-1 at the University of Iowa Hospitals and Clinics between January 1, 2008 and July 1, 2012. Eyes with visual loss due to HSV or HZV keratopathy were included in the statistical analysis. The main outcome measures were graft retention, postoperative complications, and visual outcome., Results: Nine eyes met the inclusion criteria, including 5 eyes in the HSV group and 4 eyes in the HZV group. The graft retention rate was 100% in the HSV group after a mean follow-up of 48.4 months, compared with 25% in the HZV group after 50.5 months (P = 0.048). There were 3 cases of microbial keratitis, including 2 eyes that also developed endophthalmitis, in the HZV group, compared with no cases in the HSV group (P = 0.048). There was significantly better best-corrected visual acuity at the most recent examination in the HSV group than in the HZV group (P = 0.019). All 5 HSV eyes had improved best-corrected visual acuity compared with preoperative acuity, whereas only 1 HZV eye experienced a similar result (P = 0.048)., Conclusions: Kpro-1 is associated with an excellent prognosis for graft retention, acceptably low prevalence of sight-threatening complications, and highly satisfactory visual improvement in eyes with HSV keratopathy, but not in eyes with HZV keratopathy.

Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Caveolin-1 (Cav-1), the signature protein of caveolae is expressed in several cell types in the adult retina and is linked to ocular pathologies including uveitis, diabetic retinopathy, and primary open angle glaucoma. Genetic ablation of Cav-1 causes retinal functional deficits due to disruptions in environmental homeostasis. To better understand Cav-1 function in the retina, we examined its expression/localization during postnatal retinal development. From P0-P5, Cav-1 was detected only in the developing superficial retinal vessels, in hyaloid and choroidal vasculature, and in the retinal pigment epithelium (RPE). At P7, staining began to be observed centrally in radial cells in the neuroretina, and this staining increased dramatically by P9/10 in identifiable Müller glia. Prominent vascular staining continued throughout development. These results support the idea that Cav-1 is an indicator of Müller glial differentiation and suggests that it plays an important role in Müller cell function.

Purpose: To report the outcomes of Boston keratoprosthesis (K-pro) type I implantation for congenital aniridia., Methods: A retrospective review of the medical records of every patient with congenital aniridia who underwent Boston K-pro type I implantation at the University of Iowa Hospitals and Clinics from January 1, 2009, through December 31, 2011 was performed. The main outcome measures were visual acuity, graft retention, and postoperative complications., Results: A total of 7 eyes (7 patients) met the inclusion criteria. The mean patient age was 52 years (range, 12-85 years). The preoperative visual acuity was 20/1600 in 6 eyes (85.7%) and hand motions in 1 eye (14.3%). After a median follow-up period of 18 months (range, 3-30 months), the median final best spectacle-corrected visual acuity was 20/200 (range, 20/100 to light perception). This included 2 eyes (28.6%) that were 20/100 and 5 eyes (71.4%) that were better than 20/300. Compared with the preoperative best spectacle-corrected visual acuity, the final vision was improved in 6 eyes (85.7%) and worse in 1 eye (14.3%). The K-pro graft was retained in all 7 eyes (100%). The most common complication was the formation of a retroprosthetic membrane in 3 eyes (42.9%), none of which required either a YAG capsulotomy or a vitrectomy. One eye (14.3%) developed a wound dehiscence that required surgical repair., Conclusions: The Boston K-pro type I is a good option for the visual rehabilitation of eyes with congenital aniridia.

Objective: The objective of the study was to compare the efficacy and outcomes of 2 vs 1 stitch at the time of cervical cerclage placement for preterm birth prevention., Study Design: This was a retrospective cohort study of women with singleton gestation undergoing history- or ultrasound-indicated transvaginal cervical cerclage prior to 24 weeks. The primary outcome was delivery at less than 37 weeks. The secondary outcomes included gestational age at delivery at less than 35, less than 34, less than 32, less than 28, and less than 24 weeks, perioperative details at the time of cerclage placement and removal, and maternal and neonatal outcomes. Comparison was made between patients with 2 vs 1 stitch at the time of cerclage placement. History- and ultrasound-indicated cerclages were analyzed separately., Results: Four hundred forty-four patients met inclusion criteria, 237 being history indicated (2 stitches, n = 86, 1 stitch, n = 151), and 207 ultrasound indicated (2 stitches, n = 117, 1 stitch, n = 90). Gestational age at delivery at less than 37 weeks was not significantly different between the 2 groups for both history- and ultrasound-indicated cerclage, even after adjusting for demographic differences and suture type (39% vs 35%; adjusted odds ratio, 1.38; 95% confidence interval, 0.64-3.01; and 44% vs 49%; adjusted odds ratio, 0.66; 95% confidence interval, 0.27-1.61, respectively)., Conclusion: Two stitches at the time of cerclage do not appear to improve pregnancy outcome either in the history- or the ultrasound-indicated procedures, compared with 1 stitch., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Purpose: To describe the clinical course of a patient requiring ophthalmic care for entropion and fungal keratitis in the setting of undiagnosed enophthalmos after previous ventriculoperitoneal shunting consistent with silent brain syndrome., Methods: Case report., Results: A 33-year-old man who had a history of ventriculoperitoneal shunting for an encephalocele during infancy presented because of ocular irritation and entropion, which was presumed to be entirely due to a chemical injury he had sustained 2 years before. He underwent 2 upper eyelid entropion repairs and developed fungal aspergilloma keratitis in the postoperative period. He underwent 2 penetrating keratoplasties and a limbal stem cell transplant but had complications with reinfection of the graft, eventually leading to a prephthisical painful eye. During enucleation, an aspergilloma was found within an enlarged superior fornix. Computed tomographic scan revealed severe enophthalmos with air between the lids and the globe, which was consistent with silent brain syndrome., Conclusions: In patients with silent brain syndrome, the lack of apposition between the eyelids and the globe results in entropion, trichiasis, lagophthalmos, and ocular irritation. This can complicate entropion repair and the severity of infectious keratitis. We also propose that the enlarged fornices seen in silent brain syndrome can also serve as a reservoir for infection, similar to the pathogenesis seen in the giant fornix syndrome.

U.S. Food and Drug Administration (FDA) approval of 17α-hydroxyprogesterone caproate for the indication of decreasing the risk of preterm delivery in those high-risk patients who previously had spontaneous preterm birth has come at considerable cost to the health care system. Weekly injections provided by compounding pharmacies starting at 16-20 weeks of gestation and continuing until 36 weeks currently cost the health care system $200 to $300 per pregnancy. This cost is significantly less than the costs associated with delivering and caring for preterm children. Makena, by KV Pharmaceutical, the same 17α-hydroxyprogesterone caproate product, is priced at $1,500 per injection, or a projected cost of $30,000 per pregnancy. With approximately 132,000 pregnancies being eligible for treatment annually, this increase in cost of 75-150 times what previously had been paid far exceeds the benefits derived from the FDA-approved Makena when compared with previously available compounded versions of 17α-hydroxyprogesterone caproate. This increased health care cost is not justified at this time. The price barrier to access imposed by KV Pharmaceutical actually could result in an increase in preterm deliveries over current rates. Actions are needed by the FDA, national societies, and the manufacturer to ensure that all high-risk patients continue to get the needed therapy to reduce the number of preterm births.

Background: Postpartum endometritis is usually a polymicrobial infection caused by organisms that are part of the normal vaginal flora. A pathologically confirmed case of postpartum endometritis secondary to herpes and cytomegalovirus in a human immunodeficiency virus (HIV)-positive patient is reported., Case: A 29-year-old, HIV-positive woman presented 6 days postpartum with abdominal pain and foul-smelling vaginal discharge. Pelvic ultrasonography revealed retained products of conception. Dilation and evacuation was performed, and antibiotics were started. Despite adequate antibiotics and laparoscopic drainage of a pelvic collection, fevers and pain continued. A total abdominal hysterectomy, salpingectomy, and appendectomy were performed. Pathology reported herpes and cytomegalovirus infection of the uterus., Conclusion: Herpes simplex virus (HSV) and cytomegalovirus need to be considered as a potential cause of postpartum endometritis. When antibiotic therapy fails, an antiviral regimen should be considered.

Purpose: To compare the outcome of penetrating keratoplasty (PKP) and deep anterior lamellar keratoplasty (DALK) in the surgical management of keratoconus (KC)., Patient and Methods: A retrospective review was conducted of the medical records of all patients treated with PKP or DALK for KC at University of Iowa Hospitals and Clinics from January 1, 2000, to December 31, 2006. The main outcome measures were visual outcome, graft survival, and complications. Cases with a minimum follow-up of 6 months were included in the statistical analysis., Results: Of 41 eyes that met the inclusion criteria, 30 eyes were treated with PKP and 11 eyes were treated with DALK. The mean follow-up was almost identical for eyes treated with PKP or DALK (21.9 vs. 22.5 months, respectively). At the most recent examination, the mean best spectacle-corrected visual acuity (BSCVA) was 20/28 for the PKP group and 20/29 for the DALK group (P = 0.77). The percentage of eyes that achieved BSCVA of 20/25 or better was higher in the PKP group than in the DALK group (77.3 vs. 45.5%, respectively), but this difference was not statistically significant (P = 0.72). Endothelial rejection occurred in 4 (13.3%) eyes after PKP. Visually significant interface haze occurred in the early postoperative course in 2 (18.2%) eyes after DALK. No cases of late-onset endothelial failure were found in either group., Conclusion: Treatment of KC with PKP or DALK is associated with similar visual outcomes, graft survival, and prevalence of sight-threatening complications.

Fewer than 1 in 5 patients comply with the established follow-up protocol to treat presumed ectopic pregnancy medically in an urban clinic population. Institutions should consider tracking their patient compliance with follow-up to determine the efficacy of their treatment decisions., (Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In recent years, host cell caveolae/caveolins have emerged as potentially important targets for pathogenic microorganisms; therefore, we investigated the role of caveolin-1 (Cav-1) in T. cruzi infection using Cav-1 null mice. Cav-1 null and wild type mice were infected with the virulent Tulahuen strain. The mortality was 100% in both groups, but death was slightly delayed in wild type mice. The parasitemia in the Cav-1 null mice was significantly reduced compared with wild type littermates. Histopathologic examination of the heart revealed numerous pseudocysts, myonecrosis, and marked inflammation, which was similar in both mouse groups. Real-time PCR confirmed these observations. Infection of cultured cardiac fibroblasts obtained from Cav-1 null and wild type mice revealed no differences in infectivity. Determination of serum levels of several inflammatory mediators revealed a striking reduction in IFN-gamma, TNF-alpha and components of the nitric oxide pathway in infected Cav-1 null mice. Infection of wild type mice resulted in the expected enhancement of inflammatory mediators. The defective production of chemokines and cytokines observed in vivo is in part attributed to Cav-1 null macrophages. Despite these marked differences in the response to infection by inflammatory mediators between the two mouse strains, the final outcome was similar. These results suggest that Cav-1 may play an important role in the normal development of immune responses.

Here, we examine the role of "non-muscle" caveolins (Cav-1 and Cav-2) in skeletal muscle biology. Our results indicate that skeletal muscle fibers from male Cav-1(-/-) and Cav-2(-/-) mice show striking abnormalities, such as tubular aggregates, mitochondrial proliferation/aggregation, and increased numbers of M-cadherin-positive satellite cells. Notably, these skeletal muscle defects were more pronounced with increasing age. Because Cav-2-deficient mice displayed normal expression levels of Cav-1, whereas Cav-1-null mice exhibited an almost complete deficiency in Cav-2, these skeletal muscle abnormalities seem to be due to loss of Cav-2. Thus, Cav-2(-/-) mice represent a novel animal model-and the first genetically well-defined mouse model-that can be used to study the pathogenesis of tubular aggregate formation, which remains a poorly understood age-related skeletal muscle abnormality. Finally, because Cav-1 and Cav-2 were not expressed within mature skeletal myofibers, our results indicate that development of these abnormalities probably originates in stem/precursor cells, such as satellite cells or myoblasts. Consistent with this hypothesis, skeletal muscle isolated from male Cav-3(-/-) mice did not show any of these abnormalities. As such, this is the first study linking stem cells with the genesis of these intriguing muscle defects.

A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in Salmonella pathogenesis. Cav-1(-/-) mice displayed a significant decrease in survival when challenged with Salmonella enterica serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1(-/-) mice. However, infection of Cav-1(-/-) macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1(-/-) mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1(-/-) mice were unable to control the systemic infection of Salmonella. The increased chemokine production in Cav-1(-/-) mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1(-/-) macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to Salmonella LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to S. enterica serovar Typhimurium.

The liver is an important target of Trypanosoma cruzi infection. Infection of CD-1 mice with T. cruzi (Brazil strain) resulted in parasitism of the liver, primarily in sinusoidal and Kupffer cells. Immunoblot analysis revealed activation of extra cellular signal-regulated kinase (ERK) during the acute and subacute period of infection, but p38 mitogen activated kinase (MAPK) and JNK were not activated. The activity of important cell cycle regulatory genes was also examined in the liver following infection. There was increased expression of cyclin D1, cyclin E and cyclin A as well as proliferating cell nuclear antigen (PCNA) at 45, 60 and 215 days post infection. In addition, the levels of the cyclin-dependent kinase inhibitors p27(KIP1), p21(WAF1) and the tumor suppressor p53 were increased in the livers obtained from infected mice. Quantitative PCR revealed increased abundance of mRNA for cyclins A, D1 and E. Interestingly, cyclin A and E are ordinarily not found in the adult liver. Thus infection caused a reversion to a fetal/neonatal phenotype. These data provide a molecular basis for cell proliferation in the liver following T. cruzi infection.

Lipid rafts, membrane sub-domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside G(M1) localized to a micron-scale membrane sub-domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub-domain maintenance. G(M1) segregation was maintained in live sperm incubated under non-capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of G(M1) diffusion to the post-acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, G(M1) dramatically redistributed several microns from the acrosomal sub-domain to the post-acrosomal, non-raft sub-domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid-lipid interaction model for sub-domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin-1 maintained segregation of both sterols and G(M1), arguing against a role for lipid shells surrounding caveolin-1 in sub-domain maintenance. Scanning electron microscopy of sperm freeze-dried without fixation identified cytoskeletal structures at the sub-domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of G(M1), we found that disulfide-bonded proteins played a significant role in sub-domain segregation. Together, these data provide an example of membrane sub-domains extreme in terms of size and stability of lipid segregation, and implicate a protein-based membrane compartmentation mechanism., (Copyright 2005 Wiley-Liss, Inc.)

Here, we show that a caveolin-1 (Cav-1) deficiency leads to an amplification of the adult mammary stem cell population, both in vivo and in vitro. First, the expression of two stem cell markers, Sca-1 and Keratin 6, is dramatically increased in the hyperplastic mammary ducts of Cav-1 deficient mice, suggesting that loss of Cav-1 induces the accumulation of a progenitor cell population in the mammary gland. To independently validate these results, we reconstituted mammary acini formation in vitro via a 3D Matrigel assay system--using primary cultures of mammary epithelial cells derived from WT and Cav-1 deficient mice. We show that Cav-1 null 3D epithelial structures display an intense increase in the expression of three stem cell markers, i.e., Sca-1, keratin 6 and keratin 5. Overall, we observed a 2-to-3 fold increase in the number of Cav-1 KO acini that are positive for a given stem cell marker. Also, we show that such amplification of progenitor cells has functional consequences, as demonstrated by the abnormal presence of myoepithelial cells in the hyperplastic lesions of Cav-1 deficient mammary glands. Finally, we provide evidence that hyper-activation of Wnt/beta-catenin signaling may constitute one of the down-stream mechanisms leading to mammary stem cell accumulation. The longevity and slow-dividing properties of mammary stem cells facilitates the accumulation of genetic alterations, and renders these progenitor cells the likely precursors of malignant derivatives. As such, we propose that loss of Cav-1 induces the accumulation of mammary stem cells, and that this event may be an initiating factor during mammary tumorigenesis.

Caveolin-1 (Cav-1) is the primary structural component of caveolae and is implicated in the processes of vesicular transport, cholesterol balance, transformation, and tumorigenesis. Despite an abundance of data suggesting that Cav-1 has transformation suppressor properties both in vitro and in vivo, Cav-1 is expressed at increased levels in human prostate cancer. To investigate the role of Cav-1 in prostate cancer onset and progression, we interbred Cav-1(-/-) null mice with a TRAMP (transgenic adenocarcinoma of mouse prostate) model that spontaneously develops advanced prostate cancer and metastatic disease. We found that, although the loss of Cav-1 did not affect the appearance of minimally invasive prostate cancer, its absence significantly impeded progression to highly invasive and metastatic disease. Inactivation of one (+/-) or both (-/-) alleles of Cav-1 resulted in significant reductions in prostate tumor burden, as well as decreases in regional lymph node metastases. Moreover, further examination revealed decreased metastasis to distant organs, such as the lungs, in TRAMP/Cav-1(-/-) mice. Utilizing prostate carcinoma cell lines (C1, C2, and C3) derived from TRAMP tumors, we also showed a positive correlation between Cav-1 expression and the ability of these cells to form tumors in vivo. Furthermore, down-regulation of Cav-1 expression in these cells, using a small interfering RNA approach, significantly reduced their tumorigenic and metastatic potential. Mechanistically, we showed that loss or down-regulation of Cav-1 expression results in increased apoptosis, with increased prostate apoptosis response factor-4 and PTEN levels in Cav-1(-/-) null prostate tumors. Our current findings provide the first in vivo molecular genetic evidence that Cav-1 does indeed function as a tumor promoter during prostate carcinogenesis, rather than as a tumor suppressor.

Caveolin-3 (Cav-3) is expressed predominantly in skeletal muscle fibers, where it drives caveolae formation at the muscle cell's plasma membrane. In vitro studies have suggested that Cav-3 may play a positive role in insulin signaling and energy metabolism. We directly address the in vivo metabolic consequences of genetic ablation of Cav-3 in mice as it relates to insulin action, glucose metabolism, and lipid homeostasis. At age 2 mo, Cav-3 null mice are significantly larger than wild-type mice, and display significant postprandial hyperinsulinemia, whole body insulin resistance, and whole body glucose intolerance. Studies using hyperinsulinemic-euglycemic clamps revealed that Cav-3 null mice exhibited 20% and 40% decreases in insulin-stimulated whole body glucose uptake and whole body glycogen synthesis, respectively. Whole body insulin resistance was mostly attributed to 20% and 40% decreases in insulin-stimulated glucose uptake and glucose metabolic flux in the skeletal muscle of Cav-3 null mice. In addition, insulin-mediated suppression of hepatic glucose production was significantly reduced in Cav-3 null mice, indicating hepatic insulin resistance. Insulin-stimulated glucose uptake in white adipose tissue, which does not express Cav-3, was decreased by approximately 70% in Cav-3 null mice, suggestive of an insulin-resistant state for this tissue. During fasting, Cav-3 null mice possess normal insulin receptor protein levels in their skeletal muscle. However, after 15 min of acute insulin stimulation, Cav-3 null mice show dramatically reduced levels of the insulin receptor protein, compared with wild-type mice treated identically. These results suggest that Cav-3 normally functions to increase the stability of the insulin receptor at the plasma membrane, preventing its rapid degradation, i.e., by blocking or slowing ligand-induced receptor downregulation. Thus our results demonstrate the importance of Cav-3 in regulating whole body glucose homeostasis in vivo and its possible role in the development of insulin resistance. These findings may have clinical implications for the early diagnosis and treatment of caveolinopathies.

Recently, we have shown that loss of caveolin-1 leads to marked alterations in insulin signaling and lipolysis in white adipose tissue. However, little is known about the role of caveolin-1 in brown adipose tissue (BAT), a tissue responsible for nonshivering thermogenesis. Here, we show that caveolin-1 null mice have a mildly, yet significantly, decreased resting core body temperature. To investigate this in detail, we next subjected the mice to fasting (for 24 h) or cold treatment (4 degrees C for 24 h), individually or in combination. Interestingly, caveolin-1 null mice showed markedly decreased body temperatures in response to fasting or fasting/cold treatment; however, cold treatment alone had no effect. In addition, under these conditions caveolin-1 null mice failed to show the normal increase in serum nonesterified fatty acids induced by fasting or fasting/cold treatment, suggesting that these mice are unable to liberate triglyceride stores for heat production. In accordance with these results, the triglyceride content of BAT was reduced nearly 10-fold in wild-type mice after fasting/cold treatment, but it was reduced only 3-fold in caveolin-1 null mice. Finally, electron microscopy of adipose tissue revealed dramatic perturbations in the mitochondria of caveolin-1 null interscapular brown adipocytes. Taken together, our data provide the first molecular genetic evidence that caveolin-1 plays a critical functional and structural role in the modulation of thermogenesis via an effect on lipid mobilization.

It is generally well accepted that caveolin-3 expression is muscle specific, whereas caveolin-1 and -2 are coexpressed in a variety of cell types, including adipocytes, endothelial cells, epithelial cells, and fibroblasts. Caveolin-1 and -2 are known to form functional hetero-oligomeric complexes in cells where they are coexpressed, whereas caveolin-3 forms homo-oligomeric high molecular mass complexes. Although caveolin-2 might be expected to interact in a similar manner with caveolin-3, most studies indicate that this is not the case. However, this view has recently been challenged as it has been demonstrated that caveolin-2 and -3 are coexpressed in primary cultures of cardiac myocytes, where these two proteins can be coimmunoprecipitated. Thus it remains controversial whether caveolin-2 interacts with caveolin-3. Here, we directly address the issue of caveolin isoform protein-protein interactions by means of three distinct molecular genetic approaches. First, using caveolin-1-deficient mouse embryonic fibroblasts, in which we have stably expressed caveolin-1, -2, or -3, we find that caveolin-1 interacts with caveolin-2 in this setting, whereas caveolin-3 does not, in agreement with most published observations. Next, we used a transfected L6 myoblast cell system expressing all three caveolin proteins. Surprisingly, we found that caveolin-1, -2, and -3 all coimmunoprecipitate in this cell type, suggesting that this interaction is muscle cell specific. Similar results were obtained when the skeletal muscle of caveolin-1 transgenic animals was analyzed for caveolin-1 and caveolin-3 coimmunoprecipitation. Thus we conclude that all three caveolins can interact to form a discrete hetero-oligomeric complex, but that such complex formation is clearly muscle specific.

Caveolin-1 and caveolin-3 are expressed in the mammalian heart. Mice deficient in caveolin 1 or 3 exhibit cardiac abnormalities including left ventricular hypertrophy and reduced fractional shortening. Cardiac imaging technologies such as transthoracic echocardiography and cardiac-gated magnetic resonance imaging (MRI) are effective tools for the study of left ventricular morphology and function in mice; however, there has not been widespread use of these technologies in studies of right ventricular morphology. In particular, right ventricular wall thickness has been difficult to assess using cardiac imaging technologies. We report here the use of centerline analysis of cardiac-gated MR images to more accurately determine right ventricular wall thickness in the mouse heart. Right ventricular wall thickness was evaluated in Cav-1 null, Cav-3 null and Cav-1/3 null mice, as well as wild-type control mice. Using this technique, we find that caveolin null mice exhibit significant thickening of the right ventricular wall as compared with age-matched wild-type controls. Interestingly, right ventricular wall thickening is greatest in the Cav-1/3 null mice. Furthermore, significant right ventricular wall thickening is also seen in the Cav-1 null mice. Histological analyses revealed right ventricular hypertrophy consistent with the imaging results. These studies demonstrate the utility of MRI in determining right ventricular wall thickness and underscore the severity of the right ventricular hypertrophy in caveolin null mice.

Although they were discovered more than 50 years ago, caveolae have remained enigmatic plasmalemmal organelles. With their characteristic "flasklike" shape and virtually ubiquitous tissue distribution, these interesting structures have been implicated in a wide range of cellular functions. Similar to clathrin-coated pits, caveolae function as macromolecular vesicular transporters, while their unique lipid composition classifies them as plasma membrane lipid rafts, structures enriched in a variety of signaling molecules. The caveolin proteins (caveolin-1, -2, and -3) serve as the structural components of caveolae, while also functioning as scaffolding proteins, capable of recruiting numerous signaling molecules to caveolae, as well as regulating their activity. That so many signaling molecules and signaling cascades are regulated by an interaction with the caveolins provides a paradigm by which numerous disease processes may be affected by ablation or mutation of these proteins. Indeed, studies in caveolin-deficient mice have implicated these structures in a host of human diseases, including diabetes, cancer, cardiovascular disease, atherosclerosis, pulmonary fibrosis, and a variety of degenerative muscular dystrophies. In this review, we provide an in depth summary regarding the mechanisms by which caveolae and caveolins participate in human disease processes.

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.

Tumorigenesis is a multistep process that involves a series of genetic changes or "multiple hits," leading to alterations in signaling, proliferation, immortalization, and transformation. Many of the molecular factors that govern tumor initiation and progression remain unknown. Here, we evaluate the transformation suppressor potential of caveolin-1 (Cav-1) and its ability to cooperate with a well established tumor suppressor, the INK4a locus. To study the effects of loss of caveolin-1 on cellular transformation, we established immortalized primary mouse embryonic fibroblasts (MEFs) expressing and lacking caveolin-1 by interbreeding Cav-1 (+/+) and Cav-1 (-/-) mice with INK4a (-/-) mice. Analysis of these cells reveals that loss of caveolin-1 confers a significant growth advantage, as measured via cellular proliferation and cell cycle analysis. Loss of caveolin-1 in the INK4a (-/-) genetic background results in constitutive hyperactivation of the p42/44 MAP kinase cascade, decreased expression of p21(Cip1), as well as cyclin D1 and PCNA overexpression, consistent with their hyperproliferative phenotype. Importantly, in cells lacking Cav-1 expression, transformation by activated oncogenes (H-Ras(G12V) or v-Src) results in increased tumor growth in vivo (up to >40-fold). Finally, INK4a (-/-)/Cav-1 (-/-) mice demonstrate disturbed mammary epithelial ductal morphology, with hyperplasia, increased side-branching, and fibrosis. Our results provide important new evidence for the transformation suppressor properties of Cav-1 and the first molecular genetic evidence that Cav-1 cooperates with a tumor suppressor, namely the INK4a genetic locus.

Recently, it was shown that caveolin-1 can be redirected from the cell surface to intracellular lipid droplets in a variety of cell types. Here, we directly address the role of caveolin-1 in lipid droplet formation and breakdown, showing that caveolin-1 null mice exhibit markedly attenuated lipolytic activity. Mechanistically, although the activity of protein kinase A (PKA) was greatly increased in caveolin-1 null adipocytes, the phosphorylation of perilipin was dramatically reduced, indicating that caveolin-1 may facilitate the PKA-mediated phosphorylation of perilipin. In support of this hypothesis, coimmunoprecipitation experiments revealed that treatment with a beta(3)-adrenergic receptor agonist resulted in ligand-induced complex formation between perilipin, caveolin-1, and the catalytic subunit of PKA in wild-type but not in caveolin-1 null fat pads. We also show that caveolin-1 expression is important for efficient lipid droplet formation because caveolin-1 null embryonic fibroblasts stably transfected with perilipin accumulated approximately 4.5-fold less lipid than perilipin-transfected wild-type cells. Finally, high-pressure freeze-substitution electron microscopy of adipose tissue revealed dramatic perturbations in the architecture of the "lipid droplet cortex" (the interface between the lipid droplet surface and the cytoplasm) in caveolin-1 null perigonadal adipocytes. Taken together, our data provide the first molecular genetic evidence that caveolin-1 plays a critical functional and structural role in the modulation of both lipid droplet biogenesis and metabolism in vivo.

Caveolae are 50-100 nm flask-shaped invaginations of the plasma membrane found in most cell types. Caveolin-1 is the principal protein component of caveolae membranes in nonmuscle cells. The recent development of Cav-1-deficient mice has allowed investigators to study the in vivo functional role of caveolae in the context of a whole animal model, as these mice lack morphologically detectable caveolae membrane domains. Surprisingly, Cav-1 null mice are both viable and fertile. However, it remains unknown whether loss of caveolin-1 significantly affects the overall life span of these animals. To quantitatively determine whether loss of Cav-1 gene expression confers any survival disadvantages with increasing age, we generated a large cohort of mice (n = 180), consisting of Cav-1 wild-type (+/+) (n = 53), Cav-1 heterozygous (+/-) (n = 70), and Cav-1 knockout (-/-) (n = 57) animals, and monitored their long-term survival over a 2 year period. Here, we show that Cav-1 null (-/-) mice exhibit an approximately 50% reduction in life span, with major declines in viability occurring between 27 and 65 weeks of age. However, Cav-1 heterozygous (+/-) mice did not show any changes in long-term survival, indicating that loss of both Cav-1 alleles is required to mediate a reduction in life span. Mechanistically, these dramatic reductions in life span appear to be secondary to a combination of pulmonary fibrosis, pulmonary hypertension, and cardiac hypertrophy in Cav-1 null mice. Taken together, our results provide the first demonstration that loss of Cav-1 gene expression and caveolae organelles dramatically affects the long-term survival of an organism. In addition, aged Cav-1 null mice may provide a new animal model to study the pathogenesis and treatment of progressive hypertrophic cardiomyopathy and sudden cardiac death syndrome.

Caveolae are specialized membrane microdomains present within the plasma membrane of the vast majority of cell types. They have a unique composition in that they are highly enriched in cholesterol, sphingolipids, and their coat proteins the caveolins (-1, -2, and -3). In recent years it has been recognized that caveolae act as signaling platforms, serving as a concentrating point for numerous signaling molecules, as well as regulating flux through many distinct signaling cascades. Although caveolae are found in a variety of cell types, they are most abundant in adipose tissue. This fact has led to the intense study of the function of these organelles in adipocytes. It has now become apparent that effective insulin signaling in the adipocyte may be strictly dependent on localization of at least two insulin-responsive elements to caveolae (insulin receptor and GLUT4), as well as on a direct functional interaction between caveolin-1 and the insulin receptor. We present a critical discussion of these recent findings.

Several lines of evidence suggest that a functional relationship exists between caveolin-1 and insulin signaling. However, it remains unknown whether caveolin-1 is normally required for proper insulin receptor signaling in vivo. To address this issue, we examined the status of insulin receptor signaling in caveolin-1 (-/-)-deficient (Cav-1 null) mice. Here, we show that Cav-1 null mice placed on a high-fat diet for 9 mo develop postprandial hyperinsulinemia. An insulin tolerance test (ITT) revealed that young Cav-1 null mice on a normal chow diet are significantly unresponsive to insulin, compared with their wild-type counterparts. This insulin resistance is due to a primary defect in adipose tissue, as evidenced by drastically reduced insulin receptor protein levels (>90%), without any changes in insulin receptor mRNA levels. These data suggest that caveolin-1 acts as a molecular chaperone that is necessary for the proper stabilization of the insulin receptor in adipocytes in vivo. In support of this notion, we demonstrate that recombinant expression of caveolin-1 in Cav-1 null mouse embryo fibroblasts rescues insulin receptor protein expression. These data provide evidence that the lean body phenotype observed in the Cav-1 knockout mice is due, at least in part, to a defect in insulin-regulated lipogenesis.

The myocardium of CD1 mice was examined for the activation of signal transduction pathways leading to cardiac inflammation and subsequent remodeling during Trypanosoma cruzi infection (Brazil strain). The activity of three pathways of the mitogen-activated protein kinases (MAPKs) was determined. Immunoblotting revealed a persistent elevation of phosphorylated (activated) extracellular-signal-regulated kinase (ERK), which regulates cell proliferation. During infection there was a transient activation of p38 MAPK but no activation of Jun N-terminal kinase. Early targets of activated ERK, c-Jun and c-Fos, were elevated during infection, as demonstrated by semiquantitative reverse transcription-PCR. Immunostaining revealed that the endothelium and the interstitial cells were most intensely stained with antibodies to c-Jun and c-Fos. Soon after infection, AP-1 and NF-kappa B DNA binding activity was increased. Protein levels of cyclin D1, the downstream target of ERK and NF-kappa B, were induced during acute infection. Immunostaining demonstrated increased expression of cyclin D1 in the vascular and endocardial endothelium, inflammatory cells, and the interstitial areas. Increased expression of the cyclin D1-specific phosphorylated retinoblastoma protein (Ser780) was also evident. Immunoblotting and immunostaining also demonstrated increased expression of proliferating cellular nuclear antigen that was predominantly present in the inflammatory cells, interstitial areas (i.e., fibroblasts), and endothelium. These data demonstrate that T. cruzi infection results in activation of the ERK-AP-1 pathway and NF-kappa B. Cyclin D1 expression was also increased. These observations provide a molecular basis for the activation of pathways involved in cardiac remodeling in chagasic cardiomyopathy.