Zika Virus Nonstructural Protein 1 Disrupts Glycosaminoglycans and Causes Permeability in Developing Human Placentas.

BACKGROUND: During pregnancy, the Zika flavivirus (ZIKV) infects human placentas, inducing defects in the developing fetus. The flavivirus nonstructural protein 1 (NS1) alters glycosaminoglycans on the endothelium, causing hyperpermeability in vitro and vascular leakage in vivo in a tissue-dependent manner. The contribution of ZIKV NS1 to placental dysfunction during ZIKV infection remains unknown. METHODS: We examined the effect of ZIKV NS1 on expression and release of heparan sulfate (HS), hyaluronic acid (HA), and sialic acid on human trophoblast cell lines and anchoring villous explants from first-trimester placentas infected with ZIKV ex vivo. We measured changes in permeability in trophoblasts and stromal cores using a dextran-based fluorescence assay and changes in HA receptor expression using immunofluorescent microscopy. RESULTS: ZIKV NS1 in the presence and absence of ZIKV increased the permeability of anchoring villous explants. ZIKV NS1 induced shedding of HA and HS and altered expression of CD44 and lymphatic endothelial cell HA receptor-1, HA receptors on stromal fibroblasts and Hofbauer macrophages in villous cores. Hyaluronidase was also stimulated in NS1-treated trophoblasts. CONCLUSIONS: These findings suggest that ZIKV NS1 contributes to placental dysfunction via modulation of glycosaminoglycans on trophoblasts and chorionic villi, resulting in increased permeability of human placentas.

Survey of cellular immune responses to human cytomegalovirus infection in the microenvironment of the uterine-placental interface.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, yet there are no established treatments for preventing maternal-fetal transmission. During first trimester, HCMV replicates in basal decidua that functions as a reservoir for virus and source of transmission to the attached placenta and fetal hemiallograft but also contains immune cells, including natural killer cells, macrophages, and T cell subsets, that respond to pathogens, protecting the placenta and fetus. However, the specific cellular and cytokine responses to infection are unknown, nor are the immune correlates of protection that guide development of therapeutic strategies. Here we survey immune cell phenotypes in intact explants of basal decidua infected with a clinical pathogenic HCMV strain ex vivo and identify specific changes occurring in response to infection in the tissue environment. Using 4-color immunofluorescence microscopy, we found that at 3 days postinfection, virus replicates in decidual stromal cells and epithelial cells of endometrial glands. Infected cells and effector memory CD8+ T cells (TEM) in contact with them make IFN-γ. CD8+ TEM cells produce granulysin and cluster at sites of infection in decidua and the epithelium of endometrial glands. Quantification indicated expansion of two immune cell subtypes-CD8+ TEM cells and, to a lesser extent, iNKT cells. Approximately 20% of immune cells were found in pairs in both control and infected decidua, suggesting frequent cross-talk in the microenvironment of decidua. Our findings indicate a complex immune microenvironment in basal decidua and suggest CD8+ TEM cells play a role in early responses to decidual infection in seropositive women.

Zika Virus Replicates in Proliferating Cells in Explants From First-Trimester Human Placentas, Potential Sites for Dissemination of Infection.

Background: Maternal Zika virus (ZIKV) infection with prolonged viremia leads to fetal infection and congenital Zika syndrome. Previously, we reported that ZIKV infects primary cells from human placentas and fetal membranes. Here, we studied viral replication in numerous explants of anchoring villi and basal decidua from first-trimester human placentas and midgestation amniotic epithelial cells (AmEpCs). Methods: Explants and AmEpCs were infected with American and African ZIKV strains at low multiplicities, and ZIKV proteins were visualized by immunofluorescence. Titers of infectious progeny, cell proliferation, and invasiveness were quantified. Results: In anchoring villus, ZIKV replicated reproducibly in proliferating cytotrophoblasts in proximal cell columns, dividing Hofbauer cells in villus cores, and invasive cytotrophoblasts, but frequencies differed. Cytotrophoblasts in explants infected by Nicaraguan strains were invasive, whereas those infected by prototype MR766 largely remained in cell columns, and titers varied by donor and strain. In basal decidua, ZIKV replicated in glandular epithelium, decidual cells, and immune cells. ZIKV-infected AmEpCs frequently occurred in pairs and expressed Ki67 and phosphohistone H3, indicating replication in dividing cells. Conclusions: ZIKV infection in early pregnancy could target proliferating cell column cytotrophoblasts and Hofbauer cells, amplifying infection in basal decidua and chorionic villi and enabling transplacental transmission.

Persistent Cytomegalovirus Infection in Amniotic Membranes of the Human Placenta.

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, including microcephaly, neurological deficits, hearing impairment, and vision loss. We previously reported that epithelial cells in amniotic membranes of placentas from newborns with intrauterine growth restriction and underlying congenital HCMV infection contain viral proteins in cytoplasmic vesicles. Herein, we immunostained amniotic membranes from 51 placentas from symptomatic and asymptomatic congenital infection with HCMV DNA in amniotic fluid and/or newborn saliva, intrauterine growth restriction, preterm deliveries, and controls. We consistently observed HCMV proteins in amniotic epithelial cells (AmEpCs) from infected placentas, sometimes with aberrant morphology. Primary AmEpCs isolated from mid-gestation placentas infected with pathogenic VR1814 proliferated and released infectious progeny for weeks, producing higher virus titers than late-gestation cells that varied by donor. In contrast to intact virion assembly compartments in differentiated retinal pigment epithelial cells, infected AmEpCs made dispersed multivesicular bodies. Primary AmEpCs and explants of amniochorionic membranes from mid-gestation placentas formed foci of infection, and interferon-ß production was prolonged. Infected AmEpCs up-regulated anti-apoptotic proteins survivin and Bcl-xL by mechanisms dependent and independent of the activated STAT3. Amniotic membranes naturally expressed both survivin and Bcl-xL, indicating that fetal membranes could foster persistent viral infection. Our results suggest strengthening innate immune responses and reducing viral functions could suppress HCMV infection in the fetal compartment.

Human cytomegalovirus infection interferes with the maintenance and differentiation of trophoblast progenitor cells of the human placenta.

UNLABELLED: Human cytomegalovirus (HCMV) is a major cause of birth defects that include severe neurological deficits, hearing and vision loss, and intrauterine growth restriction. Viral infection of the placenta leads to development of avascular villi, edema, and hypoxia associated with symptomatic congenital infection. Studies of primary cytotrophoblasts (CTBs) revealed that HCMV infection impedes terminal stages of differentiation and invasion by various molecular mechanisms. We recently discovered that HCMV arrests earlier stages involving development of human trophoblast progenitor cells (TBPCs), which give rise to the mature cell types of chorionic villi-syncytiotrophoblasts on the surfaces of floating villi and invasive CTBs that remodel the uterine vasculature. Here, we show that viral proteins are present in TBPCs of the chorion in cases of symptomatic congenital infection. In vitro studies revealed that HCMV replicates in continuously self-renewing TBPC lines derived from the chorion and alters expression and subcellular localization of proteins required for cell cycle progression, pluripotency, and early differentiation. In addition, treatment with a human monoclonal antibody to HCMV glycoprotein B rescues differentiation capacity, and thus, TBPCs have potential utility for evaluation of the efficacies of novel antiviral antibodies in protecting and restoring placental development. Our results suggest that HCMV replicates in TBPCs in the chorion in vivo, interfering with the earliest steps in the growth of new villi, contributing to virus transmission and impairing compensatory development. In cases of congenital infection, reduced responsiveness of the placenta to hypoxia limits the transport of substances from maternal blood and contributes to fetal growth restriction. IMPORTANCE: Human cytomegalovirus (HCMV) is a leading cause of birth defects in the United States. Congenital infection can result in permanent neurological defects, mental retardation, hearing loss, visual impairment, and pregnancy complications, including intrauterine growth restriction, preterm delivery, and stillbirth. Currently, there is neither a vaccine nor any approved treatment for congenital HCMV infection during gestation. The molecular mechanisms underlying structural deficiencies in the placenta that undermine fetal development are poorly understood. Here we report that HCMV replicates in trophoblast progenitor cells (TBPCs)-precursors of the mature placental cells, syncytiotrophoblasts and cytotrophoblasts, in chorionic villi-in clinical cases of congenital infection. Virus replication in TBPCs in vitro dysregulates key proteins required for self-renewal and differentiation and inhibits normal division and development into mature placental cells. Our findings provide insights into the underlying molecular mechanisms by which HCMV replication interferes with placental maturation and transport functions.

A high-affinity native human antibody neutralizes human cytomegalovirus infection of diverse cell types.

Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.

Intrauterine growth restriction caused by underlying congenital cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus (HCMV) is the major viral etiology of congenital infection and birth defects. Fetal transmission is high (30%-40%) in primary maternal infection, and symptomatic babies have permanent neurological, hearing, and vision defects. Recurrent infection is infrequently transmitted (2%) and largely asymptomatic. Congenital infection is also associated with intrauterine growth restriction (IUGR). METHODS: To investigate possible underlying HCMV infection in cases of idiopathic IUGR, we studied maternal and cord sera and placentas from 19 pregnancies. Anti-HCMV antibodies, hypoxia-related factors, and cmvIL-10 were measured in sera. Placental biopsy specimens were examined for viral DNA, expression of infected cell proteins, and pathology. RESULTS: Among 7 IUGR cases, we identified 2 primary and 3 recurrent HCMV infections. Virus replicated in glandular epithelium and lymphatic endothelium in the decidua, cytotrophoblasts, and smooth muscle cells in blood vessels of floating villi and the chorion. Large fibrinoids with avascular villi, edema, and inflammation were significantly increased. Detection of viral proteins in the amniotic epithelium indicated transmission in 2 cases of IUGR with primary infection and 3 asymptomatic recurrent infections. CONCLUSIONS: Congenital HCMV infection impairs placental development and functions and should be considered as an underlying cause of IUGR, regardless of virus transmission to the fetus.

Cytomegalovirus infection and antibody protection of the developing placenta.

Human cytomegalovirus (HCMV) infection is transmitted from the infected mother to the placenta and fetus. Virus replicates in the decidua, invasive cytotrophoblasts that breach the uterine vasculature and villous cytotrophoblasts underlying syncytiotrophoblasts, then reaches blood vessels in the villus core. Virus replication, fibrosis, and edema result in a hypoxic intrauterine environment and release of cytokines that stimulates compensatory development of the placenta. We employed villous explant cultures to study viral effects on differentiation and test novel approaches to rescue the placenta from infection.

Cytomegalovirus impairs cytotrophoblast-induced lymphangiogenesis and vascular remodeling in an in vivo human placentation model.

We investigated human cytomegalovirus pathogenesis by comparing infection with the low-passage, endotheliotropic strain VR1814 and the attenuated laboratory strain AD169 in human placental villi as explants in vitro and xenografts transplanted into kidney capsules of SCID mice (ie, mice with severe combined immunodeficiency). In this in vivo human placentation model, human cytotrophoblasts invade the renal parenchyma, remodel resident arteries, and induce a robust lymphangiogenic response. VR1814 replicated in villous and cell column cytotrophoblasts and reduced formation of anchoring villi in vitro. In xenografts, infected cytotrophoblasts had a severely diminished capacity to invade and remodel resident arteries. Infiltrating lymphatic endothelial cells proliferated, aggregated, and failed to form lymphatic vessels. In contrast, AD169 grew poorly in cytotrophoblasts in explants, and anchoring villi formed normally in vitro. Likewise, viral replication was impaired in xenografts, and cytotrophoblasts retained invasive capacity, but some partially remodeled blood vessels incorporated lymphatic endothelial cells and were permeable to blood. The expression of both vascular endothelial growth factor (VEGF)-C and basic fibroblast growth factor increased in VR1814-infected explants, whereas VEGF-A and soluble VEGF receptor-3 increased in those infected with AD169. Our results suggest that viral replication and paracrine factors could undermine vascular remodeling and cytotrophoblast-induced lymphangiogenesis, contributing to bleeding, hypoxia, and edema in pregnancies complicated by congenital human cytomegalovirus infection.

Predictive value of margins in diagnostic biopsies of nonmelanoma skin cancers.

BACKGROUND: With geographic regional variation, nonmelanoma skin cancer biopsy reports include assessment of margins. When margins are reported as negative, clinical dilemmas may emerge concerning the necessity of additional treatment. OBJECTIVE: To evaluate the predictive value of biopsy margins with regard to residual tumor present in subsequent excisions of nonmelanoma skin cancers. METHODS: This is a retrospective review of 235 diagnostic nonmelanoma skin cancer biopsies and their corresponding excisions for margin status at biopsy, and the presence of residual tumor in subsequent excisions. RESULTS: Twelve of 148 squamous cell carcinomas (8.1%) had negative biopsy margins and all of the subsequent excisions were free of residual tumor. The squamous cell carcinomas with negative biopsy margins consisted predominantly of nonfacial, superficial tumors of the well-differentiated and keratoacanthoma subtype. Nine of 87 basal cell carcinomas (10.3%) had negative biopsy margins. Seven of those 9 (77.8%) had residual tumor present in subsequent excisions. LIMITATIONS: Statistical analysis performed reached significance, but with small sample size as only 21 of the biopsy specimens had negative margins. Also, residual tumor was determined via standard bread-loafing technique on excisions, which is known to examine only a proportion of the tissue and can lead to false-negative results. CONCLUSIONS: The results of this small pilot study suggest that negative-margin diagnostic biopsies may be therapeutic for well-differentiated or keratoacanthoma subtypes of squamous cell carcinoma because all subsequent excisions were devoid of tumor. Negative biopsy margins from basal cell carcinomas were not predictive of tumor removal.

Neutralizing Antibodies to Human Cytomegalovirus Recombinant Proteins Reduce Infection in an Ex Vivo Model of Developing Human Placentas.

Human cytomegalovirus (HCMV) is the leading viral cause of congenital disease and permanent birth defects worldwide. Although the development of an effective vaccine is a public health priority, no vaccines are approved. Among the major antigenic targets are glycoproteins in the virion envelope, including gB, which facilitates cellular entry, and the pentameric complex (gH/gL/pUL128-131), required for the infection of specialized cell types. In this study, sera from rabbits immunized with the recombinant pentameric complex were tested for their ability to neutralize infection of epithelial cells, fibroblasts, and primary placental cell types. Sera from rhesus macaques immunized with recombinant gB or gB plus pentameric complex were tested for HCMV neutralizing activity on both cultured cells and cell column cytotrophoblasts in first-trimester chorionic villus explants. Sera from rabbits immunized with the pentameric complex potently blocked infection by pathogenic viral strains in amniotic epithelial cells and cytotrophoblasts but were less effective in fibroblasts and trophoblast progenitor cells. Sera from rhesus macaques immunized with the pentameric complex and gB more strongly reduced infection in fibroblasts, epithelial cells, and chorionic villus explants than sera from immunization with gB alone. These results suggest that the pentameric complex and gB together elicit antibodies that could have potential as prophylactic vaccine antigens.

Porcine xenograft biosynthetic wound dressings for the management of postoperative Mohs wounds.

Cadaveric allografts and a large variety of other biologic dressings have been reported as being useful for the postoperative management of Mohs micrographic surgery (MMS) wounds. Although the use of porcine xenografts for the immediate postoperative management of these wounds is known, their use has not been detailed in the dermatology literature. A case series of 15 consecutive Mohs micrographic surgery patients (mean age = 74.9 years, range = 49 to 89 years) with wounds initially managed with porcine xenografts is described. Porcine xenografts were useful in a variety of clinical settings following MMS. These included: (1) wound management when tumor margins were indeterminate pending additional dermatopathology studies and (2) wound management when there are issues such as through and through nasal defects involving the mucosa, large wound depth, exposed cartilage and or bone, or patient medical comorbidities that delay or prevent plans for immediate wound reconstruction. Future controlled studies of biologic dressings are needed to determine which options are best for micrographic surgery wounds. Comparisons should also include the traditional option of second intention healing without biologic dressings.

VEGF Maintains Maternal Vascular Space Homeostasis in the Mouse Placenta through Modulation of Trophoblast Giant Cell Functions.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that acts primarily on endothelial cells, but numerous studies suggest that VEGF also acts on non-endothelial cells, including trophoblast cells. Inhibition of VEGF signaling by excess production of the endogenous soluble VEGF receptor sFlt1 in trophoblast cells has been implicated in several pregnancy complications. Our previous studies and other reports have shown that VEGF directly regulates placental vascular development and functions and that excess VEGF production adversely affects placental vascular development. Trophoblast giant cells (TGCs) line the maternal side of the placental vasculature in mice and function like endothelial cells. In this study, we specifically examined the effect of excess VEGF signaling on TGC development associated with defective placental vascular development using two mouse models an endometrial VEGF overexpression model and a placenta-specific sFlt1 knockdown model. Placentas of endometrial VEGF-overexpressing dams at embryonic days (E) 11.5 and 14.5 showed dramatic enlargement of the venous maternal spaces in junctional zones. The size and number of the parietal TGCs that line these venous spaces in the placenta were also significantly increased. Although junctional zone venous blood spaces from control and VEGF-overexpressing dams were not markedly different in size at E17.5, the number and size of P-TGCs were both significantly increased in the placentas from VEGF-overexpressing dams. In sFlt1 knockdown placentas, however, there was a significant increase in the size of the sinusoidal TGC-lined, alkaline phosphatase-positive maternal blood spaces in the labyrinth. These results suggest that VEGF signaling plays an important role in maintaining the homeostasis of the maternal vascular space in the mouse placenta through modulation of TGC development and differentiation, similar to the effect of VEGF on endothelial cells in other vascular beds.

The imbalanced expression of matrix metalloproteinases in nephrogenic systemic fibrosis.

BACKGROUND: Nephrogenic systemic fibrosis (NSF) occurs in patients with renal dysfunction and gadolinium exposure. Although little is known about the pathogenesis of this disease, increased expression of transforming growth factor-beta has been recently demonstrated. Other fibrosing conditions have been shown to express an imbalance in matrix metalloproteinase (MMP) expression and their corresponding inhibitors. Myofibroblast differentiation, in which cells often express alpha-smooth muscle actin and achieve the ability to contract, is also a hallmark of fibrosis. OBJECTIVE: We theorized that NSF may overexpress tissue inhibitor of metalloproteinase-1 (TIMP-1), while simultaneously showing decreased expression of MMP-1. As a secondary aim, we sought to evaluate the presence of smooth muscle actin in our samples. METHODS: We applied immunohistochemistry to 16 skin biopsies from 10 patients with NSF using antibodies to TIMP-1, MMP-1, MMP-2, MMP-9, and alpha-smooth muscle actin. Samples from normal skin, scar, keloid and scleroderma were stained for comparison. RESULTS: TIMP-1 was strongly expressed in all NSF specimens compared to normal skin. MMP-1 expression was nearly absent in all tested samples. In all 16 NSF cases, the dermal spindle cells did not stain for alpha-smooth muscle actin. MMP-2 and MMP-9 expression was variable but was increased compared to normal skin. LIMITATIONS: The expression is semiquantitative and based on immunohistochemistry and unconfirmed by other techniques. CONCLUSIONS: In NSF, TIMP-1 is strongly expressed and MMP-1 is nearly absent, characteristic of the MMP imbalances seen in other fibrosing processes. Using smooth muscle actin immunohistochemistry, there was no evidence of myofibroblast differentiation.

Letter: Localized cutaneous reaction to intramuscular vitamin K in a patient with acute fatty liver of pregnancy.

Vitamin K1 is frequently used in the treatment and prevention of hypoprothrombinemia and hemorrhagic disease of the newborn. It also serves as an antidote to anticoagulants. Erythematous, indurated, pruritic plaques uncommonly occur in adults after intramuscular injection with vitamin K1. We present a case of a localized cutaneous reaction to intramuscular vitamin K1 in a peripartum patient with acute fatty liver of pregnancy. The history and clinical presentation of our case is presented with a discussion of the pathogenesis pathogenesis of vitamin K1 and available treatment for this condition.

Pathogenesis of Preeclampsia and Therapeutic Approaches Targeting the Placenta.

Preeclampsia (PE) is a serious pregnancy complication, affecting about 5-7% of pregnancies worldwide and is characterized by hypertension and damage to multiple maternal organs, primarily the liver and kidneys. PE usually begins after 20 weeks' gestation and, if left untreated, can lead to serious complications and lifelong disabilities-even death-in both the mother and the infant. As delivery is the only cure for the disease, treatment is primarily focused on the management of blood pressure and other clinical symptoms. The pathogenesis of PE is still not clear. Abnormal spiral artery remodeling, placental ischemia and a resulting increase in the circulating levels of vascular endothelial growth factor receptor-1 (VEGFR-1), also called soluble fms-like tyrosine kinase-1 (sFlt-1), are believed to be among the primary pathologies associated with PE. sFlt-1 is produced mainly in the placenta during pregnancy and acts as a decoy receptor, binding to free VEGF (VEGF-A) and placental growth factor (PlGF), resulting in the decreased bioavailability of each to target cells. Despite the pathogenic effects of increased sFlt-1 on the maternal vasculature, recent studies from our laboratory and others have strongly indicated that the increase in sFlt-1 in PE may fulfill critical protective functions in preeclamptic pregnancies. Thus, further studies on the roles of sFlt-1 in normal and preeclamptic pregnancies are warranted for the development of therapeutic strategies targeting VEGF signaling for the treatment of PE. Another impediment to the treatment of PE is the lack of suitable methods for delivery of cargo to placental cells, as PE is believed to be of placental origin and most available therapies for PE adversely impact both the mother and the fetus. The present review discusses the pathogenesis of PE, the complex role of sFlt-1 in maternal disease and fetal protection, and the recently developed placenta-targeted drug delivery system for the potential treatment of PE with candidate therapeutic agents.

Anesthetic and analgesic methods for gynecologic brachytherapy: A meta-analysis and systematic review.

Gynecologic brachytherapy procedures require targeted procedural and anesthetic needs including optimization of intra- and post-procedure analgesia, low rate of complications, and appropriate and timely transitions of care. It is uncertain whether neuraxial or general anesthesia is superior for these and other anesthetic outcomes. After a targeted search of the recent literature for anesthesia and analgesia studies for gynecologic brachytherapy, twenty studies were identified and appraised for potential review. Meta-analysis showed a decreased frequency in rescue analgesic administration in patients who underwent neuraxial anesthesia compared with general anesthesia for the procedure and literature review showed a comparable rate of anesthesia-related complications. Neuraxial anesthesia may be considered for gynecologic brachytherapy because of improved pain control, decreased opioid consumption, and similar rate of anesthesia complications.

Lymphatic invasion detected by D2-40/S-100 dual immunohistochemistry does not predict sentinel lymph node status in melanoma.

BACKGROUND: Sentinel lymph node biopsy provides important prognostic information with controversial therapeutic advantages. D2-40 is a novel immunohistochemical stain specific for lymphatic endothelium often utilized to study tumor lymphangiogenesis and lymphatic invasion. OBJECTIVE: To increase the detection of lymphatic invasion in primary cutaneous melanomas with D2-40/S-100 dual immunohistochemistry, and then apply the technique to melanomas with known sentinel lymph node status. The primary aim was to assess whether the presence or absence of lymphatic invasion could predict sentinel lymph node status. The secondary aims were to assess whether lymphatic invasion and/or sentinel lymph node involvement were associated with clinicopathologic parameters commonly studied in melanomas. METHODS: Twenty-seven biopsy specimens of primary cutaneous melanoma from 27 patients with known sentinel lymph node status were retrospectively reviewed and labeled with D2-40/S-100 dual immunohistochemistry. The following clinicopathologic variables were evaluated: age, gender, histologic type, Breslow thickness, Clark level, ulceration, mitoses, lymphovascular invasion by routine staining and D2-40/S-100 dual immunohistochemistry, and overall survival. Statistical analyses were performed to assess for associations. RESULTS: D2-40/S-100 dual immunohistochemistry showed unequivocal lymphatic invasion in 10 of 27 melanomas compared with 1 of 27 with routine histology. Eight of 10 melanomas with lymphatic invasion were sentinel lymph node negative. There was no statistical association between the presence or absence of lymphatic invasion and sentinel lymph node status. LIMITATIONS: The major limitation was the small sample size. CONCLUSION: D2-40/S-100 dual immunohistochemistry increases the sensitivity of detection of lymphatic invasion in melanoma but does not predict sentinel lymph node involvement.

Neutralizing Monoclonal Antibodies Reduce Human Cytomegalovirus Infection and Spread in Developing Placentas.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects worldwide, yet the most effective strategies for preventing virus transmission during pregnancy are unknown. We measured the efficacy of human monoclonal antibodies (mAbs) to HCMV attachment/entry factors glycoprotein B (gB) and the pentameric complex, gH/gL-pUL128-131, in preventing infection and spread of a clinical strain in primary placental cells and explants of developing anchoring villi. A total of 109 explants from five first-trimester placentas were cultured, and infection was analyzed in over 400 cell columns containing ~120,000 cytotrophoblasts (CTBs). mAbs to gB and gH/gL, 3-25 and 3-16, respectively, neutralized infection in stromal fibroblasts and trophoblast progenitor cells. mAbs to pUL128-131 of the pentameric complex, 1-103 and 2-18, neutralized infection of amniotic epithelial cells better than mAbs 3-25 and 3-16 and hyperimmune globulin. Select mAbs neutralized infection of cell column CTBs, with mAb 2-18 most effective, followed by mAb 3-25. Treatment of anchoring villi with mAbs postinfection reduced spread in CTBs and impaired formation of virion assembly compartments, with mAb 2-18 achieving better suppression at lower concentrations. These results predict that antibodies generated by HCMV vaccines or used for passive immunization have the potential to reduce transplacental transmission and congenital disease.

Nephrogenic systemic fibrosis is associated with transforming growth factor beta and Smad without evidence of renin-angiotensin system involvement.

BACKGROUND: The mechanisms of fibrosis associated with nephrogenic systemic fibrosis (NSF) are largely unknown. Transforming growth factor beta (TGF-beta), a known profibrotic cytokine, is theorized to play a central role. The renin-angiotensin system has been linked with both TGF-beta expression and fibrosis in other organ systems. OBJECTIVE: We sought to investigate whether these mechanisms were involved with NSF. METHOD: Eleven biopsy specimens from 8 patients with NSF were evaluated by immunohistochemistry for the expression of TGF-beta, Smad 2/3, angiotensin-converting enzyme (ACE), and angiotensin II receptor 1 (AT1). RESULTS: TGF-beta was detected in 8 of 11 samples of NSF. Smad 2/3 nuclear staining was seen in 8 of 11 samples. Conversely, only faint staining for ACE was seen in 2 of the 11 specimens. No AT1 staining was seen. LIMITATIONS: We did not perform our studies on a cohort of comparable patients with renal dysfunction without NSF. Our technique may not have been sufficiently sensitive to detect renin-angiotensin system involvement. CONCLUSIONS: TGF-beta, as well as its second messengers, Smad 2/3, appears to be associated with the fibrosis seen in NSF. No definitive evidence of renin-angiotensin system involvement could be determined.