Considerations for understanding protein measurements: Identification of formation, degradation and more pathological relevant epitopes.

OBJECTIVES: There is a need for precision medicine and an unspoken promise of an optimal approach for identification of the right patients for value-based medicine based on big data. However, there may be a misconception that measurement of proteins is more valuable than measurement of fewer selected biomarkers. In population-based research, variation may be somewhat eliminated by quantity. However, this fascination of numbers may limit the attention to and understanding of the single. This review highlights that protein measurements (with collagens as examples) may mean different things depending on the targeted epitope - formation or degradation of tissues, and even signaling potential of proteins. DESIGN AND METHODS: PubMed was searched for collagen, neo-epitope, biomarkers. RESULTS: Ample examples of assays with specific epitopes, either pathological such as HbA1c, or domain specific such as pro-peptides, which total protein arrays would not have identified were evident. CONCLUSIONS: We suggest that big data may be considered as the funnel of data points, in which most important parameters will be selected. If the technical precision is low or the biological accuracy is limited, and we include suboptimal quality of biomarkers, disguised as big data, we may not be able to fulfill the promise of helping patients searching for the optimal treatment. Alternatively, if the technical precision of the total protein quantification is high, but we miss the functional domains with the most considerable biological meaning, we miss the most important and valuable information of a given protein. This review highlights that measurements of the same protein in different ways may provide completely different meanings. We need to understand the pathological importance of each epitope quantified to maximize protein measurements.

Basement membrane collagen IV deficiency promotes abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) is a complex disease which is incompletely accounted for. Basement membrane (BM) Collagen IV (COL4A1/A2) is abundant in the artery wall, and several lines of evidence indicate a protective role of baseline COL4A1/A2 in AAA development. Using Col4a1/a2 hemizygous knockout mice (Col4a1/a2+/-, 129Svj background) we show that partial Col4a1/a2 deficiency augmented AAA formation. Although unchallenged aortas were morphometrically and biomechanically unaffected by genotype, explorative proteomic analyses of aortas revealed a clear reduction in BM components and contractile vascular smooth muscle cell (VSMC) proteins, suggesting a central effect of the BM in maintaining VSMCs in the contractile phenotype. These findings were translated to human arteries by showing that COL4A1/A2 correlated to BM proteins and VSMC markers in non-lesioned internal mammary arteries obtained from coronary artery bypass procedures. Moreover, in human AAA tissue, MYH11 (VSMC marker) was depleted in areas of reduced COL4 as assessed by immunohistochemistry. Finally, circulating COL4A1 degradation fragments correlated with AAA progression in the largest Danish AAA cohort, suggesting COL4A1/A2 proteolysis to be an important feature of AAA formation. In sum, we identify COL4A1/A2 as a critical regulator of VSMC phenotype and a protective factor in AAA formation.

The novel collagen matrikine, endotrophin, is associated with mortality and cardiovascular events in patients with atherosclerosis.

BACKGROUND: Rupture of atherosclerotic plaques is the major cause of acute cardiovascular events. The biomarker PRO-C6 measuring Endotrophin, a matrikine of collagen type VI, may provide valuable information detecting subjects in need of intensified strategies for secondary prevention. OBJECTIVE: In this study, we evaluate endotrophin in human atherosclerotic plaques and circulating levels of PRO-C6 in patients with atherosclerosis, to determine the predictive potential of the biomarker. METHODS: Sections from the stenotic human carotid plaques were stained with the PRO-C6 antibody. PRO-C6 was measured in serum of patients enrolled in the Carotid Plaque Imagining Project (CPIP) (discovery cohort, n = 577) and the innovative medicines initiative surrogate markers for micro- and macrovascular hard end-points for innovative diabetes tools (IMI-SUMMIT, validation cohort, n = 1,378). Median follow-up was 43 months. Kaplan-Meier curves and log-rank tests were performed in the discovery cohort. Cox proportional hazard regression analysis (HR with 95% CI) was used in the discovery cohort and binary logistic regression (OR with 95% CI) in the validation cohort. RESULTS: PRO-C6 was localized in the core and shoulder of the atherosclerotic plaque. In the discovery cohort, PRO-C6 independently predicted future cardiovascular events (HR 1.089 [95% CI 1.019 -1.164], p = 0.01), cardiovascular death (HR 1.118 [95% CI 1.008 -1.241], p = 0.04) and all-cause death (HR 1.087 [95% CI 1.008 -1.172], p = 0.03). In the validation cohort, PRO-C6 predicted future cardiovascular events (OR 1.063 [95% CI 1.011 -1.117], p = 0.017). CONCLUSION: PRO-C6 is present in the atherosclerotic plaque and associated with future cardiovascular events, cardiovascular death and all-cause mortality in two large prospective cohorts.

Can biomarkers of extracellular matrix remodelling and wound healing be used to identify high risk patients infected with SARS-CoV-2?: lessons learned from pulmonary fibrosis.

Pulmonary fibrosis has been identified as a main factor leading to pulmonary dysfunction and poor quality of life in post-recovery Severe Acute Respiratory Syndrome (SARS) survivor's consequent to SARS-Cov-2 infection. Thus there is an urgent medical need for identification of readily available biomarkers that in patients with SARS-Cov-2 infection are able to; (1) identify patients in most need of medical care prior to admittance to an intensive care unit (ICU), and; (2) identify patients post-infection at risk of developing persistent fibrosis of lungs with subsequent impaired quality of life and increased morbidity and mortality. An intense amount of research have focused on wound healing and Extracellular Matrix (ECM) remodelling of the lungs related to lung function decline in pulmonary fibrosis (PF). A range of non-invasive serological biomarkers, reflecting tissue remodelling, and fibrosis have been shown to predict risk of acute exacerbations, lung function decline and mortality in PF and other interstitial lung diseases (Sand et al. in Respir Res 19:82, 2018). We suggest that lessons learned from such PF studies of the pathological processes leading to lung function decline could be used to better identify patients infected with SARS-Co-V2 at most risk of acute deterioration or persistent fibrotic damage of the lung and could consequently be used to guide treatment decisions.

Minilaparotomic incision for haemorrhagic corpus luteum: a case report.

Corpus luteum cyst rupture with consequent hemoperitoneum is a common cause of admission to the emergency room. This condition is frequently misdiagnosed because of overlapping of clinical findings in acute gynecologic diseases. However, an incorrect identification may lead to delay in surgical treatment, which can Romabe a life-threatening condition. Ultrasound (US) is the first technique used for diagnosis that can confirm or dismiss the presence of intraperitoneal fluid. Secondly, the contrast-enhanced computed tomography (CT) is the quickest way to identify the site of active bleeding and to establish the correct management of the clinical condition. Herein, we report a case of a 19-years-old girl with acute abdominal pain correctly identified by diagnostic images and treated with mini-invasive surgery techniques in order to quickly act without clinic and aesthetic sequelae.

Exploring the role of extracellular matrix proteins to develop biomarkers of plaque vulnerability and outcome.

Cardiovascular disease (CVD) is the most common cause of death in industrialized countries. One underlying cause is atherosclerosis, which is a systemic disease characterized by plaques of retained lipids, inflammatory cells, apoptotic cells, calcium and extracellular matrix (ECM) proteins in the arterial wall. The biologic composition of an atherosclerotic plaque determines whether the plaque is more or less vulnerable, that is prone to rupture or erosion. Here, the ECM and tissue repair play an important role in plaque stability, vulnerability and progression. This review will focus on ECM remodelling in atherosclerotic plaques, with focus on how ECM biomarkers might predict plaque vulnerability and outcome.

A biomarker of collagen type I degradation is associated with cardiovascular events and mortality in patients with atherosclerosis.

OBJECTIVE: Atherosclerosis is characterized by accumulation of lipids, cells and extracellular matrix (ECM) proteins in the arterial wall. Collagen type I (COL1), a component of the arterial ECM, is cleaved by matrix metalloproteinases (MMPs) and known to be remodelled in atherosclerosis. We explored whether the MMP-mediated COL1 biomarker, C1M, was associated with cardiovascular events, cardiovascular mortality and all-cause mortality in a large prospective cohort of patients with known atherosclerosis. METHODS: Serum from 787 patients who underwent a carotid endarterectomy was included. Circulating levels of C1M were measured in serum. A total of 473 patients were followed for 6 years after surgery. Associations between C1M and incidence of cardiovascular events, cardiovascular mortality and all-cause mortality were assessed by Kaplan-Meier curves and Cox regression analysis. RESULTS: A total of 101 (21.4%) patients suffered from nonfatal cardiovascular events during the follow-up period, and 64 (13.5%) patients died. Of these, 39 (60.9%) died from cardiovascular diseases. Patients with C1M levels above the median were significantly associated with cardiovascular events, cardiovascular mortality and all-cause mortality (P < 0.001, P = 0.004 and P < 0.001, respectively). C1M was included in the final model for prediction of cardiovascular events (HR 2.15, 95% CI 1.40-3.32, P = 0.001), cardiovascular mortality (HR 2.20, 95% CI 1.07-4.51, P = 0.031) and all-cause mortality (HR 2.98 95% CI 1.67-5.33, P = < 0.001). CONCLUSIONS: In patients with atherosclerotic carotid lesions, high levels of C1M predicted cardiovascular events, cardiovascular mortality and all-cause mortality. These findings emphasize the importance of remodelling mechanisms in atherosclerosis that are now becoming more and more explored.

Diagnosis and management of fetal ductus arteriosus constriction-closure.

Pathognomonic features of in utero premature restriction/closure of the ductus arteriosus (DA) are increased right ventricular afterload, impaired right ventricular function, and consequently tricuspid regurgitation and right heart dilation. The most common reason for constriction-closure of DA is maternal administration of non-steroidal anti-inflammatory drugs (NSAIDs) during the 3rd trimester of gestation. The idiopathic form is a rare event and, maybe, an underestimated abnormality that, if it is not promptly recognized, may result in severe fetal-neonatal compromise. We describe a case of a 38-year-old woman presenting at 34+0 weeks of gestation with a normally grown male fetus whose fetal echocardiography had shown right ventricular hypertrophy, a tortuous S-shaped DA and a significant pulmonary hyperflow. All signs were consistent of an idiopathic severe constriction of DA with a significant fetal cardiac involvement. The patient was admitted to a tertiary care center equipped with Neonatal Intensive Care Unit (NICU), and delivered by cesarean section at 34+4 weeks with a good maternal and neonatal outcome. Based on our experience and a review of the Literature we propose a management algorithm to use when dealing with preterm or early term pregnancy complicated by this fetal hemodynamic malfunction.

Investigating the Robustness and Diagnostic Potential of Extracellular Matrix Remodelling Biomarkers in Alkaptonuria.

BACKGROUND AND AIM: Alkaptonuria (AKU) clinical manifestations resemble severe arthritis. The Suitability of Nitisinone in Alkaptonuria 1 (SONIA 1) study is a dose-finding trial for nitisinone treatment of AKU patients. We tested a panel of serum and urinary biomarkers reflecting extracellular matrix remodelling (ECMR) of cartilage, bone and connective tissue in SONIA 1 patients to identify non-invasive and diagnostic biomarkers of tissue turnover in AKU. METHODS: Fasted serum and urine were retrieved from 40 SONIA 1 patients and 44 healthy controls. Established biomarkers of bone remodelling (CTX-I, P1NP, OC), cartilage remodelling (CTX-II, C2M, AGNx1) and inflammation (CRPM) as well as exploratory biomarkers of ECMR (C6M, VCANM, MIM, TIM) were measured at baseline in serum and urine by means of enzyme-linked immunosorbent assays (ELISAs) or automated systems (Elecsys 2010). RESULTS: The levels of bone resorption (CTX-I) and cartilage degradation (C2M) were elevated in AKU patients as compared to controls (p > 0.0001 and p = 0.03, respectively). Also tissue inflammation (CRPM) was elevated in AKU patients (p = 0.01). In addition all four exploratory biomarkers of ECMR (C6M, VCANM, MIM, TIM) were elevated in AKU patients compared to healthy controls. CTX-II was the only biomarker to be reduced in AKU patients. TIM was the only marker that showed a higher concentration than the normal assay range in AKU patients. CONCLUSIONS: We have identified new potential biomarkers for assessment of cartilage, bone and cardiovascular remodelling in AKU and demonstrated the robustness of the assays used to measure the biomarker concentration in biological fluids.

The importance of extracellular matrix for cell function and in vivo likeness.

OBJECTIVE: Fibrotic diseases may be described as a disease of the extracellular matrix, where the balance between matrix formation and degradation has been shifted leading to an accumulation of matrix. Currently a fit for purpose model and readily available approach are adapted when doing cell cultures, which may not reflect physiology and pathophysiology optimally. The aim of this review is to draw special attention to the similarities and differences of current state of the art in vitro and ex vivo models, with special focus on the proteins, cell-cell interactions, and correct matrix composition, which may be a better representative of in vivo conditions in a disease where the extracellular matrix is the central player. METHODS: We reviewed current literature with emphasis on the role of the extracellular matrix in health and disease, different fibrotic disease models, and highlighting the importance of this when looking at translational science. CONCLUSION: To further our fibrotic research one paramount problem is to fundamentally understand the core of the disease, the production and degradation of the extracellular matrix. For a surprisingly long time the ECM has been underestimated until recently, with the discovery that the ECM may control cell phenotype through cell-matrix interactions. This highlights the need of a native ECM when investigating pathways and response to potential therapy. Clearly, both in vitro and in vivo models provide fit to purpose benefits, but in particular for the fibrosis field we may ask, do single cell cultures in monolayers recapitulate the complicated ECM environment controlling cell fate?

Laparoscopic removal of a giant paratubal cyst complicated by hydronephrosis.

Paratubal cysts represent approximately 10% of all adnexal masses. In most cases they are very small, but very few cases are reported in the literature where they exceed 15 cm of diameter. Furthermore, giant paratubal cysts complicated by bilateral hydronephrosis are unique. The Authors describe a case of a huge paratubal cyst (30 cm in diameter), in a 14 year old obese girl, treated by complete laparoscopic enucleation.

[Chronic pelvic pain in patients with endometriosis: results of laparoscopic treatment]. / Dolore pelvico cronico nell'endometriosi: risultati del trattamento laparoscopico.

AIM: The chronic pelvic pain (CPP) linked to endometriosis, relatively frequent condition in women of reproductive age, often represents the main complaint for which the patient seeks medical advice. The purpose of this prospective study was to evaluate if and to which extent systematic ablation of endometriotic lesions causes an improvement and/or disappearance of pain in patients with ascertained endometriosis and in whom the main preoperative symptom is chronic pelvic pain. METHODS: This study examined 109 patients, affected by chronic pelvic pain secondary to endometriosis, underwent laparoscopic treatment. All patients in the preoperative phase and only the 92 coming back for follow up, were asked both to fill out an anonymous questionnaire about their quality of life and to indicate on a numeric visual analogue scale (VAS) the intensity of the perceived pain. RESULTS: Overall the median of the VAS score for pain decreased from 7.5 before surgery to 2.5 at one-year postoperative-follow up, which was consistent (Wilcoxon test) with a statistically significant regression (P<0.0001) of the intensity of perceived pain (disappearance or marked reduction) in the operated patients, independently from the stage of the disease and the type of pain; also data on quality of life during work and social activity indicated a relevant improvement respectively in 82% and in 83% of patients following the laparoscopic procedure. CONCLUSION: For this reason and as suggested by the present international guidelines, when the medical therapy against pain fails and/or in the presence of an adnexal mass (chocolate cyst) or deep endometriotic lesion, it is generally correct to rely on surgical ablation of the lesions preferably by laparoscopy.

Characterization of an immunodominant epitope of small ruminant lentivirus (SRLV) nucleoprotein.

To extend and complete the epitope mapping of gag-encoded structural proteins, the immunodiagnostic potential of nucleoprotein (NP) of two different small ruminant lentivirus (SRLV) genotypes were antigenically characterized. Respective recombinant counterparts were generated and used in an enzyme-linked immunosorbent assay (ELISA) format to test a panel of sera from infected flocks. Results clearly indicate that a single linear epitope located within the C-terminal is partially cross-reactive among different SRLV genotypes and may complement multiple epitope ELISA for serological diagnosis of infection. However, in contrast to matrix and capsid antigen epitopes, which drive a genotype-specific immunoresponse, a moderate degree of variation was identified in NP independently of the genotype to which it belongs.

Polymorphous low-grade adenocarcinoma of the salivary glands: clinicopathological and immunohistochemical study of a case.

Polymorphous low-grade adenocarcinoma (PLGA) is a distinctive salivary gland neoplasm with a predilection for intraoral sites. Histological and immunohistochemical analyses were used to study a case involving the minor salivary glands of the buccal mucosa in a 68-year-old woman. Histologically, the tumor was characterized by unenca-psulated, progressing and focally-infiltrative borders and showed variable growth patterns including solid, tubular, microcystic and cribriform; papillary and papillary-cystic areas of more than focal extent were present. Cytologically the neoplasm was composed of uniform, round to cuboidal cells with bland, round to oval nuclei. Immunohistochemical analysis showed that the tumor cells were positive for cytokeratin, S-100 protein, vimentin, focally-positive for EMA and actin and negative for CEA. The significance of the papillary component and the possible relationship to other salivary gland tumors is discussed.

[Profile Orifice Shapers. A valid instrument also for the root canal preparation of short teeth: proposal for a series modification]. / Profile Orifice Shapers, strumenti validi anche per l'intera preparazione canalare di denti corti. Proposte di una modifica della serie.

BACKGROUND: The several endodontic instruments, currently used for the canal preparation with the crown-down technique, simplified only in part root canal therapy, but increased the costs. The authors examined the Profile Orefice Shapers Series, planned only for the preparation of the coronal third, and carried out a comparative analysis with other endodontic instruments currently available. METHODS: Eighty-seven teeth with a 19 mm work lenght were selected for this study. Endodontic treatment was performed using exclusively Profile Orefice Shapers under constant irrigation with 2.5% sodium hypoclorite to clean and shape the canal and System B and Obtura II to obturate the root canal system. RESULTS: Postoperative films show an adequate root canal shaping and a complete apical seal. Endodontic therapy was simplified (only 5 instruments to obtain a good canal shaping); clinical time and costs were decreased. CONCLUSIONS: The authors obtained a good shaping and apical seal using the Profile Orefice Shapers in short teeth. The quality of the root canal therapy obtainable with these instruments is confirmed by radiographs. Some modifications are suggested: increase of instruments working length, introduction of 3 extra intermediate instruments and restriction of working part diameter (1 mm) and operative procedures that lead to obtain an excellent and complete use of these instruments.

Beta 2 integrins are involved in cytokine responses to whole Gram-positive bacteria.

Proinflammatory cytokines have an important pathophysiologic role in septic shock. CD14 is involved in cytokine responses to a number of purified bacterial products, including LPS. However, little is known of monocyte receptors involved in cytokine responses to whole bacteria. To identify these receptors, human monocytes were pretreated with different mAbs and TNF-alpha was measured in culture supernatants after stimulation with whole heat-killed bacteria. Human serum and anti-CD14 Abs significantly increased and decreased, respectively, TNF-alpha responses to the Gram-negative Escherichia coli. However, neither treatment influenced responses to any of the Gram-positive bacteria tested, including group A and B streptococci, Listeria monocytogenes, and Staphylococcus aureus. Complement receptor type III (CR3 or CD18/CD11b) Abs prevented TNF-alpha release induced by heat-killed group A or B streptococci. In contrast, the same Abs had no effects when monocytes were stimulated with L. monocytogenes or S. aureus. Using either of the latter bacteria, significant inhibition of TNF-alpha release was produced by Abs to CD11c, one of the subunits of CR4. To confirm these blocking Ab data, IL-6 release was measured in CR3-, CR4-, or CD14-transfected Chinese hamster ovary cells after bacterial stimulation. Accordingly, streptococci triggered moderate IL-6 production (p < 0.05) in CR3 but not CD14 or CR4 transfectants. In contrast, L. monocytogenes and S. aureus induced IL-6 release in CR4 but not CR3 or CD14 transfectants. Collectively our data indicate that beta 2 integrins, such as CR3 and CR4, may be involved in cytokine responses to Gram-positive bacteria. Moreover, CD14 may play a more important role in responses to whole Gram-negative bacteria relative to Gram-positive ones.

Role of IL-10 in a neonatal mouse listeriosis model.

This study was undertaken to test the hypothesis that altered IL-10 production plays a role in the increased susceptibility of neonates to listeriosis. Plasma IL-10 levels were measured in neonatal and adult mice at various times after infection with Listeria monocytogenes. Relative to adults, neonatal mice had markedly increased IL-10 levels early in the course of infection with Listeria using a 90% lethal dose. Higher neonatal IL-10 responses were also observed after injecting adults and pups with equal doses of killed organisms. Splenic macrophages from neonates produced higher IL-10 levels than those of adults after in vitro stimulation with killed bacteria, confirming in vivo observations. Moreover, IL-10 blockade had differential effects in neonates and adults infected with live Listeria. In adult mice, anti-IL-10 Abs decreased bacterial burden early in the course of infection, but were no longer effective at 6 days or later after challenge. In the pups, however, the same treatment had beneficial effects both early and late during infection and resulted in increased survival. Collectively, our data suggest that an overproduction of IL-10 by macrophages may at least partially explain the increased susceptibility of neonates to listeriosis, and provide further evidence that cytokine production is different in adults and neonates.

Protective effect of tyrphostin AG-556 on shock induced by endotoxin or gram positive bacteria.

The effects of tyrphostin AG-556 (TYR), a tyrosine kinase inhibitor, were evaluated on shock induced by lipopolysaccharide (LPS) or group B streptococcus (GBS) in rats. Mortality and mean survival time were monitored. Plasma 6-keto prostaglandin F1alpha (6-keto PGF1alpha) was also measured at four hours after LPS injection. The effects of TYR on the production of 6-keto PGF1alpha thromboxane B2(TXB2) and nitrite (NO) from LPS or GBS stimulated in vitro peritoneal rat macrophage were also examined. Salmonella enteritidis LPS (12 mg/kg, i.v. ) (n=6) produced severe shock (100% mortality). Simultaneous treatment with TYR (n=6) significantly (p < 0.01) extended mean survival time and 33% of rats survived. Plasma 6-keto PGF1alpha concentrations were increased in LPS controls, whereas TYR (5 mg/kg) significantly (p < 0.05) decreased the production. Animals treated with GBS/D-galactosamine (n=9) also exhibited shock with 100% lethality and TYR again prolonged survival time (p < 0.05) with 55% of the animals surviving. To evaluate direct effects of TYR on mediator production induced by LPS or GBS, rat macrophages were stimulated with heat-killed GBS or LPS with or without TYR. Supernatants were collected at 24 h for determination of TXB2, 6-keto PGF1alpha and NO. All mediators measured were significantly increased (p < 0.05) with LPS or GBS. TYR inhibited (p < 0.05) the production of all mediators from macrophages induced by LPS or GBS. The decrease in eicosanoids was associated with a reduction of the content of cyclooxygenase-2 (COX-2) as determined by western blotting. Collectively, these results suggest that TYR ameliorates toxic shock induced by LPS or gram positive bacteria. This protection is associated with suppression of macrophage mediator production.

Effect of nitric oxide donors and nitric oxide synthase inhibitors in neonatal rat endotoxic shock.

Previous studies have shown an increased mortality in response to endotoxin in 24-hr-old neonatal rats compared with older neonates and adults. This increased susceptibility may be related to increased nitric oxide (NO) and thromboxane (TxB2) production. Twenty-four-hour-old neonatal rat pups were given either N(G)-nitro-L-arginine methyl ester (L-NAME; a nonspecific NO synthase inhibitor), S-methylthioisourea (SMT; a specific NO synthase inhibitor), or molsidomine (a NO donor) subcutaneously prior to or after an LD50 of intracardiac endotoxin. Mortality was followed for 72 hr. There was no statistically significant difference in mortality between control animals and those pretreated with L-NAME, SMT, or molsidomine. A trend toward increased mortality with nonspecific NO synthase inhibition and decreased mortality with the NO donor was noted. Splenic cells were obtained for in vitro cytokine stimulation studies. In vitro adherent splenic cell stimulation studies confirmed an increase in NO production with NO donor pretreatment and decreased production of NO with NO synthase inhibition pretreatment. There was no difference in TxB2 production with either the NO synthase inhibitor or the NO donor. In conclusion, at the several doses employed, neither nonselective or selective NO synthase inhibitors nor NO donors prevented endotoxin-induced mortality in rat neonatal shock. Although these findings do not preclude possible involvement of NO in neonatal pathophysiology, increased NO production thus does not appear to be the primary determinant of the increased susceptibility of the neonatal rat to endotoxic shock.

The effect of a tyrosine kinase inhibitor on endotoxin mortality and splenocyte mediator production in the neonatal rat.

Tyrosine kinases mediate cellular signal transduction to endotoxin. A class of tyrosine kinase inhibitors, the tyrphostins, have been shown to protect mice from endotoxin-induced lethality. Neonatal rats and mice have been shown to be uniquely susceptible to lethal endotoxic shock. In our study, the effect of a lipophilic tyrphostin, AG 556, on endotoxin-induced neonatal and adult mortality and in vitro neonatal splenic cell thromboxane (TxB2), tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production were examined. Neonatal rats (<24 h old) were administered tyrphostin (100 microg subcutaneous) 2 h before an approximate LD50 dose of Salmonella enteritidis endotoxin (.024 mg/kg/intracardiac). There was a significant decrease in mortality in the animals pretreated with 100 microg of tyrphostin (29% mortality in the treated group, n = 41 versus 53% in the vehicle control group, n = 40; p < .05). Also in adult rats tyrphostin (5 mg/kg intraperitoneal) 2 h before endotoxin (10 mg/kg intravenous) significantly improved survival (50% drug treated versus 84% in control, n = 12/group; p < .05). Adherent neonatal splenic cell mediator production of TxB2, TNF-alpha, and NO (measured by nitrite) in tyrphostin pretreated splenic cells were compared with endotoxin-stimulated splenic cells in vitro. The studies (n = 4) demonstrate an increase (p < .05) in the production of TxB2, TNF-alpha, and NO in the endotoxin- (10 microg/mL) stimulated adherent splenic cells compared with basal. Tyrphostin pretreatment (10, 20, 50 microM) produced a dose-dependent decrease (p < .05) in endotoxin-stimulated TxB2 and TNF-alpha production. NO production was not significantly reduced. In conclusion, tryphostin appears to have a protective effect on mortality in both adult and neonatal rat endotoxic shock. Tyrphostin decreased specific mediator production in stimulated neonatal cells. Thus, inhibition of signal transduction pathways of endotoxin activation by tyrosine kinase inhibition may provide an effective approach to treat endotoxic shock in the neonate.