Targeting tumor necrosis factor alpha. New drugs used to modulate inflammatory diseases.

Since its discovery, the understanding of the roles for TNF-alpha in human biology and disease has grown. Receptors for TNF are found on virtually all cell types, and many physiologic processes seem to be altered by TNF-alpha. The understanding of how TNF-alpha is involved in the pathophysiology of diseases, such as inflammatory diseases, has allowed the development of new drugs that can interfere with excess TNF-alpha and thus has allowed novel therapies for rheumatoid arthritis and Crohn's disease. As the role of TNF-alpha in other diseases becomes better understood, such TNF-alpha-modulating drugs may find further applications. In the skin, TNF-alpha is prominent cytokine that seems to be important in allergic and irritant contact dermatitis and inflammatory skin conditions. Modulating TNF-alpha activity in the skin may provide therapeutic benefits for a variety of skin conditions (Table 4). Tumor necrosis factor-alpha levels are elevated in skin lesions of psoriasis. A few reports have already suggested that etanercept and infliximab may offer a therapeutic effect in patients with psoriasis. Clinical studies evaluating the true efficacy of these drugs in psoriasis are under way. Specifically, the authors and others are involved in a double-blind, placebo-controlled study to assess the efficacy of etanercept for psoriasis. Thalidomide has been used off-label with some success to treat a number of dermatologic diseases, including several inflammatory skin conditions. Etanercept and infliximab might perhaps prove efficacious for inflammatory skin conditions as well. Finally, it is possible that drugs targeting TNF-alpha may have yet-unrecognized serious side effects. Because TNF-alpha seems to be a central cytokine in UVR-induced apoptosis, the chronic use of TNF-alpha-altering drugs might increase the risk for skin cancers. Tumor necrosis factor-alpha also plays some role in cutaneous wound healing; the effect these drugs might have on this process is also unknown at this time. Certainly, much is already [table: see text] known about TNF-alpha and how it plays many central roles. This understanding has allowed the development of useful new drugs for intractable disease. As the understanding of TNF-alpha and other cytokine biology increases, so will the number of potential therapeutic agents.

Evaluation of Schistosoma mansoni retinoid X receptor (SmRXR1 and SmRXR2) activity and tissue distribution.

Recently, we reported the identification of cDNA's encoding retinoid X receptor (RXR) homologues in Schistosoma mansoni. RXRs are known to be involved in the regulation of genes important for homeostasis and development. Previous studies indicated that SmRXR1 plays a role in the regulation of the female-specific gene, p14. Herein, we report that SmRXR2 also binds to cis-elements present in the p14 upstream region when evaluated in yeast reporter strains. SmRXR2 shows a pattern of recognition of cis-sequences present in the p14 gene upstream region different than SmRXR1. However, the SmRXR2 C (DNA binding) domain binds promiscuously in electrophoretic mobility shift assays to cis-elements of the p14 upstream region. The SmRXRs differ in their ability to activate transcription. The N-terminal A/B domain of SmRXR1 is necessary and sufficient for autonomous transcription activation function (AF) in yeast. SmRXR2 does not exhibit an equivalent autonomous AF. SmRXR1 and SmRXR2 fail to dimerize when investigated both in the yeast two-hybrid system and in immunoprecipitation experiments. In situ hybridization experiments using paraffin sections of adult worms demonstrate that SmRXR1 and SmRXR2 exhibit both common and unique cell type distribution which indicates that SmRXR1 and SmRXR2 both play a role in regulating gene expression in certain cells, yet each plays a distinct role in modulating the expression of genes in other cell types. Both SmRXR1 and SmRXR2 localize to vitelline cells. These studies provide a solid basis for improving our understanding of RXRs and their importance in female-specific gene regulation.

Nonsense-mediated decay of mRNA for the selenoprotein phospholipid hydroperoxide glutathione peroxidase is detectable in cultured cells but masked or inhibited in rat tissues.

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.

CADASIL: the dermatologic diagnosis of a neurologic disease. Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an increasingly recognized neurologic disease characterized by pathognomonic changes to the small vessels, particularly in the brain and skin. Although much has recently been written about this disease in the neuropathology literature, to our knowledge nothing has appeared in the dermatology literature. We wish to call attention to the unique role dermatologists and dermatopathologists can play in the diagnosis of this disease. We review the condition's clinical, histologic, and ultrastructural features.

Detection of human herpesvirus 8 DNA sequences in tissues and bodily fluids.

Human herpesvirus 8 (HHV-8) has been proposed as a sexually transmitted etiologic agent of Kaposi's sarcoma (KS). In this study, by use of a sensitive polymerase chain reaction assay, HHV-8 DNA was detected in the skin lesions (92%), normal skin (23%), peripheral blood mononuclear cells (PBMC) (46%), plasma (7%), saliva (37%), and semen (12%) but not stool samples from KS patients. The average number of HHV-8 copies per microgram of positive target DNA was 64, 000, 9000, 40, 33,000, and 300 for skin, PBMC, plasma, saliva, and semen samples, respectively. Only 1 non-KS donor sample, of saliva, was positive for HHV-8. Sequencing showed 5% divergence among HHV-8 strains. The data suggest that saliva may be more important than semen or stool in the sexual transmission of HHV-8. The relatively high prevalence of HHV-8 in PBMC raises the question as to why there is no evidence for bloodborne virus transmission.

At least one intron is required for the nonsense-mediated decay of triosephosphate isomerase mRNA: a possible link between nuclear splicing and cytoplasmic translation.

Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3'-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3'-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3'-most intron from pre-mRNA "marks" the 3'-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the "mark" mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5' untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.

Tumorigenesis of rat mammary epithelial cells by N-nitroso-N-methylurea in an in vitro system: characterization of the microtumors.

Chemical carcinogenesis is a lengthy process that involves the rather loosely defined stages of initiation, promotion, and progression. Several model systems of mammary carcinogenesis have been designed to elucidate the mechanisms of chemical carcinogenesis. Most of these systems have included animal models. While organ specific chemical carcinogenesis can be initiated in these systems, the subsequent stages of promotion and progression are difficult to study in detail. Investigations on in vitro carcinogenesis have shown transformation of mammalian cells in culture; the transformational event, however, is difficult to discern within the monolayer culture. We have recently reported the development of an in vitro carcinogenesis system that allows both the initiation as well as the progression of mammary cells in a collagen gel matrix culture system. The cells transformed by a chemical carcinogen develop into discernible microtumors within the three dimensions of a collagen gel culture. Isolation of these microtumors from the collagen gel and subsequent culture in monolayer has produced cells capable of colony formation in soft agar. The present study further characterizes these microtumors originated in vitro by analysis of cell growth kinetics versus parallel control cells. In addition, flow cytometric and cytogenetic studies have been performed to investigate the chromosomal stability of these cells. It was also observed that the microtumors, produced in vitro from mammary epithelial cells of an inbred strain of rats, show the ability to form tumors upon transplantation into the fat pad of syngeneic hosts.

In vitro carcinogenesis of mammary epithelial cells by N-nitroso-N-methylurea using a collagen gel matrix culture.

Carcinogenesis is a lengthy process which eventually culminates in the transformed phenotype, cancer. However, much remains to be defined about the process of transformation. In vivo models for the study of the carcinogenic process present limitations because it is not possible to detect the premalignant stages in the animals. An in vitro model, on the other hand, facilitates the study of the carcinogenic process because it enables one to dissect out the crucial events required for carcinogenesis to occur. As carcinogenesis is believed to be a multistep process; initiation, promotion, and progression, a multistep, in vitro system has been devised in our laboratory to mimic each of these stages. We have previously shown the formation of "microtumors" in collagen gels, induced by 7,12-dimethylbenz(a) anthracene. In the present study the direct acting water soluble, mammary carcinogen, N-nitroso-N-methylurea (NMU) was used for tumorigenesis of mammary epithelial cells in culture. Mammary epithelial cells from virgin Sprague-Dawley rats were propagated and exposed to single or multiple doses of NMU while growing as a monolayer in glass petri dishes (initiation). Initiation cells were then plated into a collagen gel matrix culture. Prolonged growth in the collagen gels afforded for the progression of the transformed cells into discernable microtumors in the three-dimensional matrix of the collagen. The morphology of these "tumors" was determined by histologic sections of the gels. Fewer, if any, such structures existed in the untreated gels.

The gut origin septic states in blunt multiple trauma (ISS = 40) in the ICU.

The association between support elements (ventilator days = Vd, enteral protein = EnP, number of antibiotics per day = AB/d) and the magnitude of the septic state (SSS) and its bacteriologic manifestations (bacti. log) in 66 patients with blunt multiple trauma (mean HTI-ISS = 40) over 1649 days have been studied retrospectively. SSS is measured by summing the standard deviation units of change in the septic direction for the 16 measurements taken every day in the intensive care unit. Increasing Vd is tightly associated with an increasing SSS (r = +0.52), after day 10 an increasing bacti. log (r = +0.21 to +0.32), and an increasing AB/d (r = +0.26) (all p less than 0.001, N = 1615 - 1626). The independent variables that best predicted Vd were delayed operations (DORS), day of rising EnP, and total positive blood cultures (TPC) (adj. R sq. = 0.84, F = 104, dF = 3/59). An increasing AB/d was associated with an increasing SSS (r = +0.38), increasing Vd (r = +0.26), and an increased bacti. log (r = +0.14 to +0.18) (all p less than 0.001, N = 1615). Only an increased EnP was consistently associated with a reduced SSS (r = -0.38) and a reduction in bacti. log (r = -0.10 to -0.21) (all p less than 0.001, N = 1626-1636). The independent variables Vd, EnP, AB/d, and TPC best predicted SSS for all surviving patients (adj. R sq. = 0.42, F = 268, dF = 4/1496). The patients who died of sepsis were not different in terms of bacti. log from those with equal Vd but were distinguished by zero EnP, high AB/d, and persistent ventilatory support. In conclusion, DORS is tightly associated with increased Vd, SSS, AB/d, and zero EnP. If Vd exceeds 10, there is an increasing bacti. log and evidence of infection probably from the gut. This responds only to increased EnP and not to AB/d. Death due to sepsis is not associated with increased bacti. log but with zero EnP and high AB/d and their consequences.

Blunt multiple trauma (ISS 36), femur traction, and the pulmonary failure-septic state.

Fifty-six blunt multiple trauma patients (HTI-ISS 22-57) were studied for the effects of immediate versus delayed internal fixation of a femur or acetabular fracture on the pulmonary failure septic state. The pulmonary failure septic state may be defined as an alveolar arterial oxygen tension difference greater than 100, plus fever and leukocytosis. These patients were divided into four groups. Group I (N = 20) had immediate internal fixation, postoperative ventilatory support, and was sitting up at 30 hours. Group II (N = 20) had 10 days of femur traction and postoperative ventilatory support. Group III (N = 9) was immediately extubated after surgery and had 30 days of femur traction. Group IV (N = 7) had special circumstances that should increase the duration of the pulmonary failure septic state. These four groups of patients were statistically identical by 20 different criteria on admission except that Group I had more recognized chest injuries than Group II (12 vs. 9). Group I required 3.4 +/- 2.6 days of ventilator support and 7.5 +/- 3.8 intensive care unit (ICU) days; they had 12 +/- 8.8 elevated white counts, 3.8 +/- 4 febrile days, 0.05 positive blood cultures per patient, four fracture complications out of 93 fractures, 59 injections of narcotics, and 23 +/- 8.6 acute care days. Ten days of femur traction doubled the duration of the pulmonary failure septic state relative to Group I at a statistically significant level for nine out of 10 criteria, while increasing the number of positive blood cultures by a factor of 10, the number of fracture complications by a factor of 3.5, and the use of injectable narcotics by a factor of 2. Thirty days of femur traction increased the duration of the pulmonary failure septic state relative to Group I by a factor of 3 to 5 for all criteria at a statistically significant level, while increasing fracture complications by a factor of 17, positive blood cultures by a factor of 74, and the use of narcotics by a factor of 2. Group IV, which had four out of seven immediate internal fixations, behaved similarly to Group II. Femoral shaft traction should be avoided in the blunt multiple trauma patients because it greatly increases the cost of care and the risk of multiple systems organ failure.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary open reduction and internal fixation of open fractures.

Our experience with primary open reduction with rigid internal fixation of 50 open fractures is presented. Twenty-seven patients had associated major multiple trauma. Twenty fractures were articular and 26 involved 3rd-degree wounds. The infection rate of 4%. A system of staged sequential debridement and wound management is presented. The authors believe this system has contributed to the low infection rate. No secondary amputations occurred. Eighty-two per cent of the patients had good functional results using Charnley's criteria. Fatal post-traumatic cardiopulmonary failure did not occur. The authors feel that early definitive fracture care employing rigid fixation which avoids casts, and allows improved wound management and early mobilization of the multiple-trauma patient, has decreased the cardiopulmonary and metabolic consequences commonly associated with polytrauma patient care.