Sustained high influenza vaccination rates and decreased safety concerns among pregnant women during the 2010-2011 influenza season.

OBJECTIVE: Intense efforts to vaccinate pregnant women against 2009 H1N1 influenza resulted in much higher vaccine uptake than previously reported. We surveyed postpartum women to determine whether high vaccination rates were sustained during the 2010-11 influenza season. METHODS: We performed cross-sectional surveys of postpartum women delivering at our institution during February-April 2010 and February-March 2011. The surveys ascertained maternal characteristics, history of influenza vaccination, and reasons for lack of vaccination. RESULTS: During the 2010-11 season, 165 (55%) of 300 women surveyed reported receiving influenza vaccination, compared to 191 of 307 (62%) during 2009-10 (p=0.08). Vaccination by an obstetrical provider was common, but decreased compared to 2009-10 (60% vs. 71%, p=0.04). While most women (76%) in 2010-11 reported that their provider recommended influenza vaccination, significantly more reported lack of discussion about vaccination (24% vs. 11%, p<0.01) compared to 2009-10. Vaccine safety concerns were cited by most (66%) women declining vaccination during 2009-10 but only 27% of women who declined in 2010-11. CONCLUSION: The vaccination rate among pregnant women at our institution was relatively sustained, although fewer providers appear to be discussing influenza vaccination in pregnancy. Concern about vaccine safety, the primary barrier during 2009-10, was much less prominent.

Oncogenic mutations regulate tumor microenvironment through induction of growth factors and angiogenic mediators.

Activating mutations in the tyrosine kinase domain of HER2 (ErbB2) have been identified in human cancers. Compared with wild-type HER2, mutant HER2 shows constitutively activate kinase activity and increased oncogenicity. Cells transformed by mutant HER2 are resistant to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and exhibit an attenuated response to the HER2 antibody trastuzumab. We investigated herein pathways through which mutant HER2 alters the extracellular environment, potentially leading to drug resistance and the effect of simultaneously targeting HER2 and the tumor cell microenvironment with a therapeutic intent. Expression of mutant HER2 in mammary epithelial cells activated autocrine transforming growth factor (TGF) beta1 signaling through a mechanism involving Rac1 and c-Jun N-terminal kinase-activating protein 1-dependent transcription. Cells transformed by an activating mutant of H-Ras (G12V) also expressed higher TGF-beta1 level through Rac1 activation. In addition, mutant HER2 induced the EGFR ligands TGF-alpha and amphiregulin at the mRNA and protein levels. Vascular endothelial growth factor, a target of the TGF-beta-Smad transcriptional regulation, was also induced as a result of expression of mutant HER2. Inhibition of TGF-beta signaling with the Alk5 small molecule inhibitor LY2109761 reduced growth and invasiveness of cells expressing mutant HER2. Combined inhibition of intracellular and paracrine effects of mutant HER2 by trastuzumab and the EGFR antibody cetuximab were more efficient than single-agent therapies. These data suggest that mutations in oncogenes such as HER2 and Ras not only alter intracellular signaling but also influence on other components of the tumor microenvironment by inducing several pro-invasive growth factors. In turn, these serve as extracellular targets of novel therapeutic strategies directed at both cancer-driving oncogenes and the modified tumor microenvironment.

Chronic myeloid leukemia-associated membranoproliferative glomerulonephritis that responded to imatinib mesylate therapy.

There is no known clinical association between chronic myelogenous leukemia (CML) and membranoproliferative glomerulonephritis (MPGN). We present a patient who was followed in the renal clinic for proteinuria of unknown etiology (3.2 g/24 h) and normal renal function who was diagnosed with CML as well as MPGN and acute renal failure at the same time. The patient's renal function and proteinuria improved when his CML was treated with imatinib mesylate, suggesting that CML either caused or exacerbated existing MGPN. To the best of our knowledge, this is the first reported case of MPGN associated with CML that improved with imatinib mesylate therapy.

Glomerular injury is exacerbated in diabetic integrin alpha1-null mice.

Excessive glomerular collagen IV and reactive oxygen species (ROS) production are key factors in the development of diabetic nephropathy. Integrin alpha1beta1, the major collagen IV receptor, dowregulates collagen IV and ROS production, suggesting this integrin might determine the severity of diabetic nephropathy. To test this possibility, wild-type and integrin alpha1-null mice were rendered diabetic with streptozotocin (STZ) (100 mg/kg single intraperitoneal injection), after which glomerular filtration rate (GFR), glomerular collagen deposition, and glomerular basement membrane (GBM) thickening were evaluated. In addition, ROS and collagen IV production by mesangial cells as well as their proliferation was measured in vitro. Diabetic alpha1-null mice developed worse renal disease than diabetic wild-type mice. A significant increase in GFR was evident in the alpha1-null mice at 6 weeks after the STZ injection; it started to decrease by week 24 and reached levels of non-diabetic mice by week 36. In contrast, GFR only increased in wild-type mice at week 12 and its elevation persisted throughout the study. Diabetic mutant mice also showed increased glomerular deposition of collagen IV and GBM thickening compared to diabetic wild-type mice. Primary alpha1-null mesangial cells exposed to high glucose produced more ROS than wild-type cells, which led to decreased proliferation and increased collagen IV synthesis, thus mimicking the in vivo finding. In conclusion, this study suggests that lack of integrin alpha1beta1 exacerbates the glomerular injury in a mouse model of diabetes by modulating GFR, ROS production, cell proliferation, and collagen deposition.

Activated type I TGFbeta receptor kinase enhances the survival of mammary epithelial cells and accelerates tumor progression.

We have examined the effects of transforming growth factor-beta (TGFbeta) signaling on mammary epithelial cell survival. Transgenic mice expressing an active mutant of Alk5 in the mammary gland (MMTV-Alk5(T204D)) exhibited reduced apoptosis in terminal endbuds and during postlactational involution. Transgene-expressing mammary cells contained lower Smad2/3 and higher c-myc levels than controls, high ligand-independent phosphatidylinositol-3 kinase (PI3K) and Akt activities, and were insensitive to TGFbeta-mediated growth arrest. Treatment with a proteasome inhibitor increased Smad2/3 levels and ligand-independent Smad transcriptional reporter activity, as well as reduced both c-myc protein and basal cell proliferation. Treatment with an Alk5 kinase small-molecule inhibitor upregulated Smad2/3 levels, reduced PI3K activity, P-Akt, and c-myc, and inhibited cell survival. Although Alk5(T204D)-expressing mice did not develop mammary tumors, bigenic MMTV-Alk(T204D) x Neu mice developed cancers that were more metastatic than those occurring in MMTV-Neu transgenics. These data suggest that (1) TGFbeta can signal to PI3K/Akt and enhance mammary epithelial cell survival in vivo before cytological or histological evidence of transformation, and (2) TGFbeta signaling can provide epithelial cells with a 'gain-of-function' effect that synergizes with oncogene-induced transformation.

Integrin beta 1 signaling is necessary for transforming growth factor-beta activation of p38MAPK and epithelial plasticity.

Transforming growth factor-beta (TGF-beta) can induce epithelial to mesenchymal transdifferentiation (EMT) in mammary epithelial cells. TGF-beta-mediated EMT involves the stimulation of a number of signaling pathways by the sequential binding of the type II and type I serine/threonine kinase receptors, respectively. Integrins comprise a family of heterodimeric extracellular matrix receptors that mediate cell adhesion and intracellular signaling, hence making them crucial for EMT progression. In light of substantial evidence indicating TGF-beta regulation of various beta(1) integrins and their extracellular matrix ligands, we examined the cross-talk between the TGF-beta and integrin signal transduction pathways. Using an inducible system for the expression of a cytoplasmically truncated dominant negative TGF-beta type II receptor, we blocked TGF-beta-mediated growth inhibition, transcriptional activation, and EMT progression. Dominant negative TGF-beta type II receptor expression inhibited TGF-beta signaling to the SMAD and AKT pathways, but did not block TGF-beta-mediated p38MAPK activation. Interestingly, blocking integrin beta(1) function inhibited TGF-beta-mediated p38MAPK activation and EMT progression. Limiting p38MAPK activity through the expression of a dominant negative-p38MAPK also blocked TGF-beta-mediated EMT. In summary, TGF-beta-mediated p38MAPK activation is dependent on functional integrin beta(1), and p38MAPK activity is required but is not sufficient to induce EMT.

Distinct domains of CD98hc regulate integrins and amino acid transport.

CD98 is a cell surface heterodimer formed by the covalent linkage of CD98 heavy chain (CD98hc) with several different light chains to form amino acid transporters. CD98hc also binds specifically to the integrin beta(1A) cytoplasmic domain and regulates integrin function. In this study, we examined the relationship between the ability of CD98hc to stimulate amino acid transport and to affect integrin function. By constructing chimeras with CD98hc and a type II transmembrane protein (CD69), we found that the cytoplasmic and transmembrane domains of CD98hc are required for its effects on integrin function, while the extracellular domain is required for stimulation of isoleucine transport. Consequently, the capacity to promote amino acid transport is not required for CD98hc's effect on integrin function. Furthermore, a mutant of CD98hc that lacks its integrin binding site can still promote increased isoleucine transport. Thus, these two functions of CD98hc are separable and require distinct domains of the protein.

Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development.

Branching morphogenesis of the ureteric bud (UB) [induced by the metanephric mesenchyme (MM)] is necessary for normal kidney development. The role of integrins in this complex developmental process is not well understood. However, the recent advent of in vitro model systems to study branching of UB cells and isolated UB tissue makes possible a more detailed analysis of the integrins involved. We detected integrin subunits alpha3, alpha6, beta1, and beta4 in both the UB and cells derived from the early UB. Blocking the function of each of these integrin subunits individually markedly inhibited branching morphogenesis in cell culture models. However, inhibiting individual integrin function with blocking antibodies in whole kidney and isolated UB culture only partially inhibited UB branching morphogenesis, suggesting that, in these more complex in vitro systems, multiple integrins are involved in the branching program. In whole organ and isolated bud culture, marked retardation of UB branching was observed only when both alpha3 and alpha6 integrin subunits were inhibited. The alpha6 integrin subunit can be expressed as both alpha6beta1 and alpha6beta4, and both of these beta subunits are important for UB branching morphogenesis in both cell and organ culture. Furthermore, laminin-5, a common ligand for integrins alpha3beta1 and alpha6beta4, was detected in the developing UB and shown to be required for normal UB branching morphogenesis in whole embryonic kidney organ culture as well as isolated UB culture. Together, these data from UB cell culture, organ culture, and isolated UB culture models indicate that both integrin alpha3 and alpha6 subunits play a direct role in UB branching morphogenesis, as opposed to being modulators of the inductive effects of mesenchyme on UB development. Furthermore the data are consistent with a role for laminin-5, acting through its alpha3beta1 and/or alpha6beta4 integrin receptors, in UB branching during nephrogenesis. These data may help to partially explain the renal phenotype seen in integrin alpha3 and alpha3/alpha6 subunit-deficient animals.

Class- and splice variant-specific association of CD98 with integrin beta cytoplasmic domains.

CD98 is a type II transmembrane protein involved in neutral and basic amino acid transport and in cell fusion events. CD98 was implicated in the function of integrin adhesion receptors by its capacity to reverse suppression of integrin activation by isolated integrin beta(1A) domains. Here we report that CD98 associates with integrin beta cytoplasmic domains with a unique integrin class and splice variant specificity. In particular, CD98 interacted with the ubiquitous beta(1A) but not the muscle-specific splice variant, beta(1D), or leukocyte-specific beta(7) cytoplasmic domains. The ability of CD98 to associate with integrin cytoplasmic domains correlated with its capacity to reverse suppression of integrin activation. The association of CD98 with integrin beta(1A) cytoplasmic domains may regulate the function and localization of these membrane proteins.

The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation.

The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.

ROS stimulate reorganization of mesangial cell-collagen gels by tyrosine kinase signaling.

Reactive oxygen species (ROS) initiate multiple pathological and physiological cellular responses, including tyrosine phosphorylation of proteins. In this study, we investigated the effects of ROS on cell-extracellular matrix interactions utilizing the floating three-dimensional collagen gel assay. Exposure of mesangial cells grown in three-dimensional culture to H2O2, 3-amino-1,2,4-triazole (a catalase inhibitor), or puromycin is associated with gel reorganization accompanied by tyrosine phosphorylation of multiple proteins, including focal adhesion kinase (FAK). Neutrophils cocultured with mesangial cells in three-dimensional culture also induce mesangial cell-collagen gel reorganization and initiate tyrosine phosphorylation of a similar set of proteins. Collectively, these results show that ROS of either endogenous or exogenous origin can modulate mesangial cell-extracellular matrix interactions through initiation of a phosphotyrosine kinase signaling cascade. Consequently, ROS may play a role as signaling molecules that regulate mesangial cell-extracellular matrix interactions in both physiological and pathological conditions.

Tyrosine kinase cell signaling pathways of rat mesangial cells in 3-dimensional cultures: response to fetal bovine serum and platelet-derived growth factor-BB.

Cells grown in 3-dimensional collagen gels adopt a nonproliferative, contractile phenotype which is more characteristic of cells in vivo than cells grown in 2-dimensional culture. The floating collagen gel contraction assay is a well-defined system used to study cell-extracellular matrix interactions grown in 3-dimensional culture. Although the cell biology of this system is well defined, the cell signaling associated with gel contraction has not been well characterized. In this study we demonstrate that fetal bovine (FBS) and platelet-derived growth factor (PDGF)-induced mesangial cell-collagen gel contraction is associated with increased tyrosine phosphorylation of a number of proteins including focal adhesion kinase (FAK) and the 42-kDa isoform of MAPK (ERK2). FBS-induced gel contraction is not affected by the presence of the MEK inhibitor PD098059. Low concentrations of PDGF-BB (10 ng/ml) induce gel contraction; however, at higher PDGF-BB concentrations (80 ng/ml) gel contraction is not observed. PDGF-BB-induced gel contraction as well as tyrosine phosphorylation of FAK are inhibited in the presence of the PI-3 kinase inhibitor wortmanin. Minimal autophosphorylation of the PDGF-beta receptor is observed under 3-dimensional culture conditions following PDGF-BB stimulation; however, when mesangial cells grown in 2-dimensional culture are exposed to PDGF-BB, the PDGF-beta receptor was prominently phosphorylated. We conclude that induction of collagen gel contraction by FBS and PDGF-BB is associated with tyrosine kinase phosphorylation and that these responses differ substantially from what occurs in 2-dimensional cultures in the presence of the same agonists.

Thrombotic microangiopathy in renal transplant recipients treated with cyclosporin A.

Thrombotic microangiopathy is an uncommon but well described complication of renal transplantation. This study is a review of the case records of 18 patients with biopsy proven post transplant thrombotic microangiopathy, without cellular rejection. There was no single characteristic underlying cause of renal failure in native kidneys. Although only two (11%) patients had undergone previous transplantation, 16 (89%) had panel reactive antibodies (PRA). All patients received prophylactic antilymphocyte globulin, a single patient had cyclosporin A (CSA) at the time of transplant and in 16 patients CSA was introduced when graft function was established. On this protocol 16 (89%) patients had early graft function. All patients developed acute renal failure and 16 (89%) required dialysis. Nine (50%) patients developed hematological abnormalities. All patients were treated aggressively with anti-rejection therapy, CSA was temporarily withdrawn, and 2 (11%) patients received plasmapheresis. Seven (39%) patients lost their grafts. Renal function in the remaining patients recovered to serum creatinine levels ranging from 104 mumol/l to 430 mumol/l (1.2 mg% to 4.8 mg%). All patients with surviving grafts had CSA successfully reintroduced. This study indicates that there is an association between patients who develop posttransplant thrombotic microangiopathy after CSA administration and high PRA levels. The condition appears to respond to anti-rejection therapy and stopping CSA in the majority of cases. The safe reintroduction of CSA suggests that endothelial cell damage in the posttransplant period may be multifactorial and not solely due to CSA therapy.

Idiopathic membranous nephropathy in the elderly: a comparative study.

This is a retrospective study of 74 elderly patients (60 years of age or older) with idiopathic membranous nephropathy. They represented 23% of the total of 323 cases of idiopathic membranous nephropathy who presented during the 19-year review period. The mean age of these patients was 67 years versus 41 in the younger-onset group. The median presenting serum creatinine in the elderly group was higher (1.3 mg/dL v 1.0 mg/dL, P < 0.001), and the median creatinine clearance calculated by Cockcroft-Gault to correct for age, gender, and weight was lower (55 mL/min v 95 mL/min, P < 0.0001). The incidence of chronic renal insufficiency, defined as a creatinine clearance of less than 50 mL/min, was significantly worse in the elderly after a mean observation period of 47 months (59% v 25%, P < 0.0001) although end-stage renal failure (ESRF) was not (18% v 12%). The rate of change of renal function, however, as measured by the time to doubling of baseline creatinine, was similar in both groups, as was the complete remission rate. Forty-six percent of patients (33 of 74) in the elderly group received treatment: steroids alone (76%), immunosuppression drugs alone (9%), or a combination (15%). There was no benefit noted in terms of complete remission rate or incidence of chronic renal insufficiency. In summary, more elderly patients with membranous nephropathy develop chronic renal insufficiency, but this appears to be related to their age and decreased functional reserve, because the rate of decline in renal function after initiation of the disease is no different than in the younger age-group. The data also indicate that an accurate assessment of renal function in this older age-group requires an estimated or calculated creatinine clearance, given the inaccuracy when only serum creatinine is used. There was no evidence of improved outcome with prednisone therapy, and in view of the increased incidence of complications associated with this drug in the elderly as well as the decreased reserve at presentation, we suggest that routine steroid treatment of these patients should not be undertaken.

Puromycin aminonucleoside inhibits mesangial cell-induced contraction of collagen gels by stimulating production of reactive oxygen species.

Glomerular epithelial cell injury is thought to be the primary reason for the development of proteinuria in puromycin aminonucleoside nephrosis (PAN), the rat model of nephrotic syndrome. By comparison mesangial cells are considered resistant to the effects of puromycin. The purpose of the present study was to investigate whether puromycin in non cytotoxic concentrations caused mesangial cell dysfunction, with particular reference to cell-extracellular matrix interactions. Mesangial cells, when embedded in collagen gels, contact after exposure to minimal essential medium (MEM) containing fetal bovine serum (FBS). This contractility, measured by determining changes in area of the collagen gel, is inhibited by puromycin in a dose dependent manner from 2.5 micrograms/ml to 160 micrograms/ml. At these concentrations there is no alteration of cell viability as measured by the tetrazolium salt (MTT) method and trypan blue exclusion. Immunocytochemistry with rhodamine phalloidin reveals that actin filaments are not disrupted. The antioxidants, superoxide dismutase (SOD) and catalase as well as diphenylene iodonium (DPI), a flavoprotein inhibitor, not only counteracted the effect of puromycin on gel contraction, but also enhanced gel contraction when added to mesangial cells on their own. Aminotriazole, an inhibitor of endogenous catalase, inhibited mesangial cell-induced gel contraction in a dose dependent manner (5 mM to 40 mM), and this effect was completely reversed by addition of catalase. Mesangial cells preloaded with dihydrorhodamine and exposed to puromycin (5 micrograms/ml to 160 micrograms/ml) exhibited a dose dependent increase in rhodamine 123 fluorescence, indicating production of reactive oxygen species (ROS). This effect was blocked by the addition of DPI.(ABSTRACT TRUNCATED AT 250 WORDS)

IgG heavy-chain deposition disease.

A 51-yr-old man presented with renal failure, proteinuria, hematuria, and hypertension. The serum contained two monoclonal protein spikes, an IgG4 lambda and free lambda light chains. Free gamma heavy chains were absent from serum. A bone marrow biopsy did not show evidence of a plasmalymphocytic dyscrasia. Renal biopsy showed a nodular glomerulopathy with linear deposits of gamma 4 heavy chains alone along glomerular, tubular, and vascular basement membranes and in mesangial regions. No light chains were detected in the kidney despite staining with antisera directed against both free and bound light chains and the F(ab')2 fragment of IgG. The term heavy-chain deposition disease is appropriate in view of the above features.

Crescentic nephritis at Groote Schuur Hospital, South Africa--not a benign disease.

The etiology of crescentic nephritis (CN) in the developing world differs from that of Europe and North America. This retrospective study of 73 patients is the largest series of CN in the developing world. The records of all renal biopsies performed at Groote Schuur Hospital, Cape Town, South Africa, over thirteen years, between January 1977 and April 1991, were reviewed. Specimens selected for this study had six or more glomeruli and over 50% of these glomeruli had crescent formation. It confirms that post infectious glomerulonephritis (PIGN) (n = 21) is the commonest cause of CN in this setting. In addition there were 15 patients with CN associated with systemic lupus erythematosus (SLE). These two groups make this study unique as they are the largest series of each described in the literature. Thirty-nine (53%) patients in this series progressed to end-stage renal failure (ESRF) and only nine (12%) patients recovered renal function to a normal serum creatinine. Eight (38%) patients in the PIGN group developed ESRF, indicating the poor prognosis of this condition. Six of eight patients in the PIGN group treated with steroids and cyclophosphamide recovered to a serum creatinine level less than 200 mumol/l and only one progressed to ESRF, which may indicate that this form of therapy is beneficial. Thirteen (87%) patients with SLE either developed ESRF or died which suggests that the presence of crescents in this condition is associated with a poor prognosis.

Diagnosis of lymphoma in a continuous ambulatory peritoneal dialysis patient by peritoneal fluid cytology.

A 72-year-old woman with diabetic nephropathy receiving continuous ambulatory peritoneal dialysis for 1 year presented with recurrent episodes of sterile cloudy dialysate. Abdominal ultrasound and computed tomography demonstrated retroperitoneal masses. Cytology of the dialysis fluid revealed large atypical lymphocytes with light chain restriction, strongly suggestive of lymphoma. Fine-needle aspiration of the retroperitoneal masses showed cells with identical cytologic and immunocytochemical staining characteristics. This is the first report of de novo lymphoma diagnosed in a continuous ambulatory peritoneal dialysis patient by cytologic analysis of the dialysis effluent.