Showing papers by "Eric G. Neilson published in 1996"

KAP-1, a novel corepressor for the highly conserved KRAB repression domain.

Abstract: The KRAB repression domain is one of the most widely distributed transcriptional effector domains yet identified, but its mechanism of repression is unknown. We have cloned a corepressor, KAP-1, which associates with the KRAB domain but not with KRAB mutants that have lost repression activity. KAP-1 can enhance KRAB-mediated repression and is a repressor when directly tethered to DNA. KAP-1 contains a RING finger, B boxes, and a PHD finger; the RING-B1-B2 structure is required for KRAB binding and corepression. We propose that KAP-1 may be a universal corepressor for the large family of KRAB domain-containing transcription factors.

Regulation of Fas and Fas ligand expression in cultured murine renal cells and in the kidney during endotoxemia

Abstract: Fas ligand (FasL) and Fas belong to a recently described family of cytokines and receptors with similarities to tumor necrosis factor (TNF) and its receptors. Upon engagement by specific antibodies or by FasL, Fas transduces a signal for apoptosis in permissive cells. Although apoptosis occurs during renal development and following injury to mature cells, the factors responsible for programmed renal cell death are uncertain. We have studied Fas expression by renal cells in vitro and during endotoxemia in mice. Several renal cell types, including glomerular mesangial cells and tubular epithelial cells express a Fas transcript in culture. Lipopolysaccharides (LPS), interleukin-1 beta, interferon-gamma (IFN-gamma), and TNF-alpha increase the levels of Fas mRNA in cultured mesangial and tubular cells. TNF-alpha and LPS raise the level of Fas mRNA in a time- and dose-dependent manner with Fas receptor expression peaking after 72 h of exposure to LPS. Anti-Fas antibodies can induce the death of cultured mesangial cells. This cell death shows the characteristic changes of apoptosis, including DNA fragmentation and pyknotic changes of the nucleus. Increases in Fas by LPS, TNF-alpha, and IFN-gamma enhance the killing induced by the anti-Fas antibody. FasL is also expressed by cultured renal cells, and TNF-alpha treatment of mesangial cells increases its expression. In vivo, Fas mRNA is present at low level in normal kidney. LPS increases the levels of Fas mRNA and protein in kidney and produces evidence of apoptosis along nephrons. These data suggest that transcripts encoding natural FasL and Fas are induced by LPS and may play a role in endotoxemia-induced acute renal failure and organ dysfunction.

From converting enzyme inhibition to angiotensin II receptor blockade: new insight on angiotensin II receptor subtypes in the kidney.

Abstract: Since the discovery of renin, almost 100 years ago, all the components of the renin-angiotensin system have been characterized on a molecular level. Angiotensin II (Ang II) receptors can be grouped in two classes: AT1 and AT2 receptors. In addition to these receptor subtypes, several other Ang-II-binding sites with different properties have been identified. The majority of Ang II receptors in the kidney comprises the AT1 type, whereas AT2 receptors are only expressed by distinct structures such as the renal capsule and the juxtaglomerular apparatus. The vast majority of physiological functions of angiotensin II in the kidney are mediated through AT1 receptors. Important exceptions may be the pressure-induced natriuresis and chemotaxis for macrophages/ monocytes, as well as growth-suppressive effects which may be mediated by AT2 receptors.

Anti-α1(IV) collagen autoantibodies associated with lung adenocarcinoma presenting as the goodpasture syndrome

Abstract: We report a case of adenocarcinoma of the lung with hemoptysis that was mistaken for the Goodpasture syndrome on the basis of a serologic test for antiglomerular basement membrane antibodies. Antiglomerular basement membrane antibodies are characteristic of the Goodpasture syndrome, some forms of rapidly progressive glomerulonephritis, and isolated instances of pulmonary hemorrhage. Antiglomerular basement membrane antibodies from patients with traditional Goodpasture syndrome react with the 3(IV) chain, one of the six known chains of type IV collagen that form an integrative lattice called basement membrane [1, 2]. Evaluation of the antiglomerular basement membrane antibodies in our patient showed the presence of anti- 1(IV) rather than anti- 3(IV) reactivity. Case History A 72-year-old man (AG) presented with recurrent hemoptysis and abnormal chest radiograph. His work-up had previously been thought to be negative with the exception of an elevated titer for antiglomerular basement membrane antibodies. His medical history was remarkable for obstructive pulmonary disease, previously treated tuberculosis, and a 90 pack-year history of cigarette smoking. Physical examination on admission showed mild respiratory distress. Laboratory findings included a hemoglobin level of 107 g/L, a serum creatinine level of 70.72 mol/L, and an erythrocyte sedimentation rate of 20 mm/h. The urinalysis result was normal. Test results for antinuclear antibodies and antineutrophil cytoplasmic antibodies were negative. The patient's chest radiograph showed right middle and right upper lobe infiltrates. Fiberoptic bronchoscopy and thoracoscopy showed diffuse hemorrhage from the right upper and right middle lobes. Wedge biopsy specimens from both lobes were consistent with acute and chronic pulmonary hemorrhage; centrolobular emphysema was seen, and vasculitis was not. Cytologic studies, bacterial cultures, acid-fast bacilli stains, and immunofluorescent studies for tissue-bound antibodies were negative; no malignancy was noted. Because a commercial reference laboratory reported an antiglomerular basement membrane antibody level of 44 (normal less than 10), limited Goodpasture syndrome was suspected. The patient was treated with steroids, cyclophosphamide, and several courses of plasmapheresis. He improved clinically until hospital day 26, when a right pleural effusion developed. Thoracentesis yielded 1 L of bloody fluid. Cytologic examination of the fluid showed an adenocarcinoma; the patient died soon thereafter. Methods Bovine type IV collagens, their hexamers, and recombinant noncollagenous domain 1 (NC1) domains for human 1- 6 type IV collagens were purified before use [3]. Immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and immunofluorescence were done using standard protocols [2, 4, 5]. Results Our patient (AG) was initially treated for the Goodpasture syndrome because the commercial serologic data and chest radiograph suggested a life-threatening autoimmune disease, despite the absence of nephritis [1]. Several cases of pulmonary hemorrhage due to antiglomerular basement membrane antibodies in the absence of renal involvement have been reported [6, 7]. To address the possibility that AG may have had such a case, we assayed plasmapheresis fluid from the first treatment for anti- 3(IV) NC1 antibodies using both native bovine and recombinant human antigens. The results of ELISA and immunoblotting showed minimal or no binding to the 3(IV) NC1 (Figure 1; top and bottom, respectively); instead, intense reactivity with both bovine 1(IV) NC1 and human recombinant 1(IV) NC1 was seen. We also noted weak binding to the human recombinant 3(IV) NC1 and 6(IV) NC1 (Figure 1, middle). Serial serum samples were further studied in retrospect using an antiglomerular basement membrane antibody radioimmunoassay [5] and were found to be nonreactive. The lung biopsy specimen from AG showed no endogenous IgG bound to alveolar basement membrane (Figure 2, left). Indirect immunofluorescence on renal tissue showed that AG's antibodies were bound to the mesangial matrix, tubular basement membrane, and glomerular basement membrane. This anti- 1(IV) NC1 antibody binding was most intense in the mesangial matrix and glomerular basement membrane (Figure 2, middle). The reference Goodpasture sera containing 3(IV) NC1 antibodies (LL) bound predominantly to the glomerular basement membrane in a linear manner [2] (Figure 2, right). The binding of AG's antibody to the mesangial matrix is characteristic of anti- 1(IV) antibodies. Figure 1. Identification of the type IV collagen chains that bind to the patient's (AG) autoantibodies. Top. Middle. Bottom. Figure 2. Tissue immunohistochemistry. Left. Middle. Right. Discussion Although the patient's antibodies were directed at antigens present in normal tissue, no inflammation was seen; therefore, we doubt that the hemoptysis was caused by immune reaction. Recent research provides some confirmation of this. Rabbits injected with bovine 1(IV) NC1 dimers produce circulating antibodies to 1(IV) NC1 domains but do not develop nephritis [4]. In contrast, when they are injected with 3(IV) NC1 dimers, rabbits develop a fulminant Goodpasture-like syndrome. These results suggest that the target glomerular basement membrane antigen may determine the likelihood of tissue damage. Our patient received three courses of five plasmaphereses. Despite the elimination of anti- 1(IV) NC1 reactivity, the patient continued to have hemoptysis, perhaps because of an erosive endobronchial tumor that was not apparent on bronchoscopy. Several reports have shown basement membrane degradation in metastatic cancer [8, 9]. Increased amounts of type IV collagen have also been seen in the sera of patients with primary or metastatic bone and soft-tissue tumors [10]. These antibodies may reflect paraneoplastic immune responses against the 1(IV) collagen chain, as seen in our patient. We believe that a definitive diagnosis of the Goodpasture syndrome requires the strong presence of anti- 3(IV) NC1 antibodies in patient serum. Although antibodies against 1(IV) NC1 are occasionally present in small amounts in a few patients with the Goodpasture syndrome, we have found that they are always associated with antibodies directed to 3(IV) NC1 domains [2]. From a clinical perspective, sensitive and specific assays using immunoabsorbed antigens [5], bovine 3(IV) NC1 chains, or recombinant human 3(IV) NC1 chains [3] are needed to accurately diagnose the Goodpasture syndrome. Drs. Palevsky and Grippi: Pulmonary Division, Department of Medicine, Hospital of the University of Pennsylvania, 422 Curie Boulevard, Philadelphia, PA 19104.

Specificity of circulating and tissue-bound autoantibodies in Goodpasture syndrome.

Abstract: Goodpasture syndrome is an often fatal autoimmune disease associated with glomerulonephritis and/or pulmonary hemorrhage. The clinical manifestations of this disease correlate well with the presence of circulating antiglomerular basement membrane (GBM) autoantibodies. The primary target antigen in glomerular and alveolar basement membranes is thought to be the alpha 3 chain of type IV collagen. Nearly all that is known about anti-GBM antibodies in humans comes from work on unbound circulating antibody. We recently had the unique and rare opportunity to obtain early postmortem antibody and tissues from a patient who died with catastrophic Goodpasture syndrome. The specificity of circulating, kidney-bound and lung-bound autoantibodies from this patient was evaluated against a variety of purified basement membrane constituents. The results indicate that the primary target for the circulating and tissue-bound autoantibodies is the NC1 domain of the alpha 3(IV) chain of type IV collagen. Additionally, all the antibodies recognize a cryptic epitope/s on the alpha 3(IV)NC1 hexamer. Furthermore, tissue-bound and circulating antibodies compete with one another for overlapping epitopes on the antigen. These findings demonstrate that circulating autoantibodies in Goodpasture syndrome are highly representative of those bound to organ tissues, strengthening the notion that pathogenic autoantibodies are targeted to the alpha 3(IV)NC1 collagen, and that previous reports of findings in the circulation may be applicable to tissue injury.

Transforming growth factor-beta modulation of the alpha 1(IV) collagen gene in murine proximal tubular cells.

Abstract: We have examined the expression of the alpha 1(IV) collagen gene in murine proximal tubular cells (MCT) to better understand how it is regulated in parenchymal cells. Transcriptional activity was examined using luciferase reporters driven by the alpha 1(IV) promoter and varying lengths of 59-flanking sequences. The minimal bidirectional promoter showed low intrinsic activity in MCT cells, but addition of upstream sequences increased luciferase expression. Maximal activity resided within the first 1,200 bp upstream. A minigene construct was generated by placing a portion of the alpha 1(IV) first intron downstream from the promoter region. The intronic sequences significantly decreased activity of the promoter in MCT cells and 3T3 fibroblasts but greatly enhanced expression in murine parietal yolk sac (PYS) endodermal cells. Addition of transforming growth factor-beta (TGF-beta) to MCT cultures elevated the levels of secreted type IV collagen. Treatment of either transiently or stably transfected MCT cells with TGF-beta produced an increase in the levels of expression of all of the reporters tested. These data support the hypothesis that cell-specific regulation of alpha 1(IV) collagen is dependent upon downstream sequences, which act to decrease the expression of type IV collagen in tubular epithelium. The activity of the alpha 1(IV) collagen gene in proximal tubular cells is increased by TGF-beta, which acts on the domain(s) embedded within the intergenic bidirectional promoter.

When does a generalist need a specialist

Abstract: T he growing work load for generalists reminds me of wha t used to happen when I went to my aunt ' s house for dinner. Every time I thought I was in control of my plate, there she 'd be with more food, and still more. If I didn ' t push away, I would stagger from the table. These days, many general is ts feel the same way about going to work, where everyone asks them to do more. The problem is worsened by several factors: the can-do mental i ty of today's primary care disciplines; t raining programs that offer too little exposure to a rapidly expanding curr iculum; the colonial a t t i tudes of professional organizations trying to s take out new ground for their members; and of course, pressure from managed care organizations when they ask their gatekeepers, \"You can handle this one more thing, can ' t you?\" Already, one ~can see concern in the faces of our trainees as they confront these issues. These increased responsibil i t ies have created a dil emma for generalists. They enjoy their prominent, new roles, bu t can they find the courage to say, \"I don' t know enough to do everything competently-\"? Somet imes this s t a tement needs to be said to a managed care network, somet imes to professional organizations represent ing the generalists ' interests, and somet imes even to patients. Awareness of one's professional l imitations always has been a critical safety ne t protecting patients, and this self-knowledge often guides how we practice. How odd it is tha t we never get a round to deselecting our incompetencies. But the silent accumula t ion of extended responsibility and the failure to keep truly up-to-date with all claimed competencies will lead to the eventual undoing of pr imary care. One consequence of the rapacious increase in generalists ' responsibili t ies will be an increase in testing for board-certif ication and recertification, with increased difficulty mainta in ing high pass rates. Another consequence will be menta l fatigue; yet another will be the temptat ion to become a \"specialized\" generalist, which is a cur ious wrinkle in internal medicine. The specialized generalist is an endrun around certification because it relies on untes ted knowledge. It is part icularly problematic for the pat ient whose illness fails within a generalist 's self-designated area of interest, because of the weakness tha t s tems from a lack of full specialty training. This brings us to the spirometry paper by Hnat iuk and colleagues in this issue of JGIM. ~ These authors found that the interpretat ion of screening spirometry by internis ts was incorrect one-third of the time. Many of the internis ts were very accurate while a few others were very inaccurate. Most of the errors were at t r ibuted to the internists not knowing publ ished guidelines. How should we interpret this information? First, it is impor tant to recognize tha t el'fort-dependent inaccuracy and technical irreproducibili ty make screening spirometry inherent ly problematic. More importantly, one should ask whether these pat ients needed this test. Would internists perform this test routinely if they couldn ' t be paid for it? Would pulmonary specialists, ff left to their own devices, want this test performed on most of the pat ients in this s tudy? My guess is \"no\" to both questions. So, wha t happens to a pat ient in today's mixed-payer marketplace who really needs spirometry?. Who should order and interpret this test? My guess is that it should be the physician who possesses the strongest competence, which on average will be the specialist. We are not the first people to th ink about these issues. Recently, while leafmg through the first edition of Cecil's Textbook ofMeclicine, 21 was s t ruck by Cecil's prescience when he explained why the textbook needed more than one author: \" . . . the rapid growth of medical science during the last few years has made it a lmost impossible for a single individual to mas te r the entire field. In internal medicine, as in other branches of h u m a n knowledge, the age of special ism has of necessi ty arrived, and some of our ablest practi t ioners even devote themselves in great measure to one disease . . . . \" Cecil wrote this in 1927 and I doubt tha t he was j u s t too lazy to write the whole book himself. It is hard to believe that, 70 years later, many in primary care medicine still do not fully realize how m u c h information they really need to know to mainta in acceptable s tandards of competence. Some don' t want to admit it because it is not politically correct or it is not in their self-interest to do so. Although generalists need specialists, the decision about when to refer pat ients is a complex one. Between 1 and 12% of primary care visits in this country resul t in referral to a specialist, a Referral ra tes are higher in group practices, among those not board-certified, and among those with less experience. 4 Referrals will always be with us because it is intuitive to make use of the best knowledge for patient care. In the era of managed care, referrals to specialists should be made more affordable, not less available. One opportuni ty to improve the relat ionship between generalists and specialists is the development of new, dec is ion-support software to help generalists make better referrals. The Consultation Guide Project star ted by Dr. Paul Ladenson at J o h n s Hopkins University is one such example. The Guide will allow a microcomputer to offer a comprehensive set of recommendat ions using existing practice guidelines, authori tat ive literature, and the judgmen t of nat ional experts. Advice is provided about the indications for referral, the t iming of referral, wha t testing should be done before referral, the need for prospective authorization, and the follow-up required after referral. Algorithms such as this one will be useful in case management, and they also may be valuable when advising pat ients who are contemplat ing premature self-referral. Finally, what do pat ients want? Culliton suggests that pat ients are fairly uninformed about their doctors '

Compositions and methods for inhibiting fibrogenesis

Abstract: Vectors, or naked DNA, containing a promoter for an FSP1 gene and a downstream gene capable of attenuating fibroblasts and their function are provided. Methods of using these vectors to inhibit tissue injury related to fibrogenesis are also provided.