Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

Direct cardiac and vascular actions of adrenomedullin in conscious sheep

1. Adrenomedullin (ADM) is a recently characterized circulating hormone which affects haemodynamic, renal and pituitary function in mammals. We have shown previously that in sheep, ADM produces vasodilatation together with increases in cardiac output and contractility. However, whether these effects are direct or mediated by autonomic reflexes is unclear. The present study examined the cardiovascular actions of an intravenous infusion of ADM in conscious, chronically instrumented sheep with either sympathetic, parasympathetic or autonomic ganglion blockade, to determine the role of the autonomic nervous system in mediating these cardiovascular changes.
2. Human ADM (1–52) was infused for 60 min at 2 μg kg⁻¹ h⁻¹ following: (1) saline control, (2) combined α/β-adrenoceptor (sympathetic) blockade (proporanolol 0.4 mg kg⁻¹ h⁻¹+phentolamine 0.15 mg kg⁻¹ h⁻¹ for 20 h), (3) muscarinic (parasympathetic) blockade (methscopolamine 0.05 mg kg⁻¹ h⁻¹ for 20 h) or (4) ganglion blockade (hexamethonium 3 mg kg⁻¹ h⁻¹ for 4 h). Measurements were made of mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), stroke volume (SV), total peripheral conductance (TPC), maximal aortic flow (Fmax) and maximal rate of change of aortic flow (dF/dt).
3. ADM reduced MAP by 3±1 mmHg, and increased CO (1.2±0.2 l min⁻¹), HR (14±2 beats min⁻¹), TPC (21±3 ml min⁻¹ mmHg⁻¹), Fmax (2.3±0.8 l min⁻¹) and dF/dt (86±21 l min⁻¹ s⁻¹) in normal sheep. In animals with α/β blockade, similar changes were observed with ADM. However, during muscarinic blockade, the increases in HR (32±4 beats min⁻¹), CO (2.1±0.4 l min⁻¹), TPC (31±4 ml min⁻¹ mmHg⁻¹), Fmax (4.0±0.6 l min⁻¹), and dF/dt (150±12 l min⁻¹ s⁻¹) produced by ADM were enhanced. During ganglion blockade, ADM produced a greater reduction in MAP (−10±2 mmHg) compared to controls (−3±1 mmHg). However, there was no increase in HR. The changes in CO, TPC and contractility were similar to those observed in control animals.
4. These results suggest that the vasodilator effects of ADM on the periphery and its ability to increase CO and cardiac contractility are not mediated by the autonomic nervous system, but are probably the result of direct actions of ADM on the heart and vasculature.

     

Motorneurons, reactive oxygen, and life span in Drosophila

Aging and life span are widely recognized, but poorly understood, aspects of basic biology. Fortunately, genetic approaches to understanding the mechanisms governing these processes are beginning to bear fruit. One line of investigation has established that incompletely reduced forms of oxygen, arising as by-products of respiration and cellular catabolism, play an important, and perhaps universal, role in aging and life span determination. An important refinement of this model of aging, suggested by recent experiments in our laboratory, is that the critical nexus of the relationship between reactive oxygen species and life span is highly localized and, in fact, may reside principally in the motorneuron. Here we analyze the strengths and weaknesses of the reactive oxygen species/motorneuron model of aging by comparing the studies on which it is based, which used the approach of targeted transgene expression in Drosophila, with studies from other laboratories using different genetic approaches, principally mutation and selection. The results encourage the view that an understanding of the mechanisms that underlie this widely recognized aspect of basic biology is within our grasp.     

A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma

We examined the relationship between a functional polymorphism (667C-- >T, ala-->val) of the methylenetetrahydrofolate reductase gene (MTHFR) and the risk of colorectal adenomas in the prospective Nurses' Health Study. Among 257 incident polyp cases and 713 controls, the MTHFR val/val polymorphism [relative risk (RR) = 1.35, 95% confidence interval (CI) 0.84-2.17] was not significantly associated with risk of adenomas. This lack of association was observed for both small (RR = 1.36, 95% CI 0.76-2.45) and large (RR = 1.32, 95% CI 0.66-2.66) adenomas. Furthermore, there was no significant interaction between this polymorphism and consumption of either folate, methionine or alcohol. We also examined the relationship of a newly identified polymorphism (asp919gly) of the methionine synthase gene (MS) with the risk of colorectal adenomas in the same population. The MS gly/gly polymorphism was also not significantly associated with risk of colorectal adenomas (RR = 0.66, 95% CI 0.26-1.70). These results, which need to be confirmed in other studies, suggest that the MTHFR val/val polymorphism, which has been previously inversely associated with risk of colorectal cancer, plays a role only in a late stage (adenoma-- >carcinoma) of colorectal tumorigenesis, and/or may protect against malignant transformation in the subset of benign adenomas, which may progress to malignancy.      

Reduction of endothelial injury after hypothermic lung preservation by initial leukocyte-depleted reperfusion

OBJECTIVES: Leukocyte depletion has been shown to ameliorate the effects of reperfusion injury in many organ systems. The aim of this study was to investigate the effects of leukocyte depletion on functional and endothelial markers of pulmonary performance after cold ischemic injury. METHOD: Groups of 6 rat lungs were flushed with University of Wisconsin solution and then stored at 4 degrees C for 4 hours. They then underwent sanguine reperfusion for 30 minutes, during which time functional measures (gas exchange, pulmonary artery, and airway pressures) were made and after which the lungs underwent estimation of endothelial permeability by measurement of the capillary filtration coefficient (in grams per centimeter of water per minute per grams of wet lung tissue) by a gravimetric technique. Four groups were studied: group 1 underwent no reperfusion, group 2 underwent 30 minutes of reperfusion, group 3 underwent 30 minutes of leukocyte-deplete reperfusion with an in-line leukocyte filter (PALL), and group 4 underwent 10 minutes of leukocyte-depleting reperfusion followed by 20 minutes of normal reperfusion. RESULTS: The capillary filtration coefficient increased between group 1 and group 2 animals (1.05 +/- 0.32 to 3.07 +/- 0.47 [mean +/- SEM]; P <.01). Complete leukocyte depletion caused the greatest diminution in the capillary filtration coefficient (0.392 +/- 0.07, P <.001), but initial leukocyte depletion (group 4) also showed a significant diminution (0.74 +/- 0.3, P <.01). Complete or initial leukocyte depletion caused no significant change in functional measures of pulmonary performance. Complete leukocyte depletion produced less pulmonary leukostasis, as assessed by means of myeloperoxidase activity. CONCLUSION: Initial and continued leukocyte depletion are associated with amelioration of reperfusion-induced endothelial injury after cold ischemic injury.     

Postpartum Thyroiditis

Post partum thyroiditis occurs in 50% of TPO AB+ve women and is characterised by transient hyperthyroidism followed by transient hypothyroidism during the first six months, post partum. A third of the latter group develop permanent hypothyroidism. The syndrome is seen in 5-9% of women and post partum thyroid dysfunction (PPTD) reoccurs in 75% of women in a subsequent pregnancy. An increase in depressive symptomatology is seen in women with PPTD as well as in ante TPO Ab+ve women without PPTD. The immunology of PPT is associated with the presence of TPO antiboides with those IgG subclasses best able to activate the complement cascade. The HLA-DR frequencies seen in PPT suggest that PPT may be related to Hashimoto's thyroiditis. TPO Ab driven complement fixation is seen in PPT and complement activation relates to the extent and progression of thyroid damage. Recent studies have shown an increase in both Th2 and Th1 cytokine release from lymphocytes in ante partum women destined to develop PPTD. More data are required on the cellular immune changes both ante partum and post partum in PPT.     

Late-onset epilepsy and occult cerebrovascular disease

The interface between cerebrovascular disease (CVD) and epilepsy is complex and multifaceted. Late-onset epilepsy (LOE) is increasingly common and is often attributed to CVD, and is indeed associated with an increased risk of stroke. This relationship is easily recognizable where there is a history of stroke, particularly involving the cerebral cortex. However, the relationship with otherwise occult, subcortical CVD is currently less well established yet causality is often invoked. In this review, we consider the diagnosis of LOE in clinical practice—including its behaviour as a potential mimic of acute ischemic stroke and transient ischemic attack; evidence for an association between occult CVD and LOE; and potential mechanisms of epileptogenesis in occult CVD, including potential interrelationships between disordered cerebral metabolism and perfusion, disrupted neurovascular unit integrity, blood–brain barrier dysfunction, and inflammation. We also discuss recently recognized issues concerning antiepileptic drug treatment and vascular risk and consider a variety of less common CVD entities associated with seizures.     

Transforming growth factor beta (TGF-beta), a potent inhibitor of erythropoiesis: neutralizing TGF-beta antibodies show erythropoietin as a potent stimulator of murine burst-forming unit erythroid colony formation in the absence of a burst-promoting activity

Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF- beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4- , and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum-containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF- beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF- beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors.     

Fluorescent in vivo tracking of hematopoietic cells. Part I. Technical considerations

We report a new technology for in vivo tracking of hematopoietic cells, using fluorescent lipophilic probes. Because the probe is irreversibly bound in the lipids of the cell membrane; substantial numbers of dye molecules can be incorporated per cell and thus substantial signal to noise can be achieved. Although this technology can be used for all hematopoietic cells, these first findings are reported on red blood cells (RBCs) owing to the importance of the membrane to RBC function and integrity. We demonstrated that labeling 10% of the RBCs of a rabbit and reinjecting them into the animal makes possible the tracking of these cells at various times after injection. Furthermore, the labeling appears not to affect in vivo cell lifetime or cellular volume changes in response to hypotonic shock. The single cell fluorescence intensity of the labeled RBCs remains relatively constant for 60 days, and an immune response appears not to be generated against labeled cells. That labeled RBCs have lifetime kinetics in vivo, as shown in other studies, indicates that the membranes are functioning normally and are unaltered by the labeling technology. The technology we present is also applicable to white blood cells, bone marrow, and platelets.