C-reactive protein-to-lymphocyte ratio is a reliable marker in patients with COVID-19 infection: the CLEAR COVID study.

OBJECTIVE: COVID-19 infection is characterized with elevation of inflammatory markers in bloodstream. A novel inflammatory marker, C-reactive protein (CRP)-to-lymphocyte ratio (CLR), is suggested to be associated with inflammation. We aimed to compare the CLR values of the deceased COVID-19 patients to the CLR of survived subjects. MATERIALS AND METHODS: The patients with COVID-19 whom presented to outpatient or inpatient clinics of AbantIzzet Baysal University Hospital were enrolled to the present retrospective study. Subjects were grouped as either deceased or survived. CLR values of the groups were compared. RESULTS: Study cohort was consisted of 568 subjects in deceased and 4753 patients in survived group. Median CLR of the deceased and survived groups were 90 (0.2-1679)% and 11 (0.2-1062)%, respectively (p < 0.001). The sensitivity (75%) and specificity (70%) of CLR (> 23.4% level) in detecting mortality were higher than those of CRP and ferritin (AUC 0.80, p < 0.001, 95% CI 0.78-0.82). CONCLUSION: We suggest that elevated CLR levels in COVID-19 patients on admission should alert physicians for poor outcome.

OBJETIVO: La infección por Covid-19 se caracteriza por elevación de marcadores inflamatorios en el torrente sanguíneo. Se sugiere que un nuevo marcador inflamatorio, la proporción de C-reactive protein (CRP) a linfocitos (CLR), está asociado con la inflamación. Nuestro objetivo fue comparar los valores de CLR de los pacientes fallecidos con Covid-19 con el CLR de los sujetos sobrevivientes. MATERIALES Y MÉTODOS: Los pacientes con Covid-19 que se presentaron en clínicas ambulatorias o de hospitalización del Hospital Universitario Abant Izzet Baysal se inscribieron en el presente estudio retrospectivo. Los sujetos se agruparon como fallecidos o sobrevivientes. Se compararon los valores de CLR de los grupos. RESULTADOS: La cohorte del estudio estuvo compuesta por 568 sujetos en el grupo fallecido y 4753 pacientes en el grupo sobreviviente. La mediana de CLR de los grupos fallecidos y sobrevivientes fue 90 (0.2-1679)% y 11 (0.2-1062)%, respectivamente (p < 0.001). La sensibilidad (75%) y la especificidad (70%) de CLR (nivel > 23.4%) en la detección de mortalidad fueron superiores a las de CRP y ferritina (AUC 0.80, p < 0.001, IC 95%: 0,78-0.82). CONCLUSIÓN: Sugerimos que los niveles elevados de CLR en pacientes con Covid-19 al ingreso deberían alertar a los médicos sobre un resultado deficiente.

Association Between Thyroid Hormone Status and Complete Blood Count Parameters in Anemic Patients

@#Introduction: Anemia and thyroid conditions effect each other in clinical practice. Anemia may induce alteration in thyroid hormone status and various thyroid conditions induce various types of anemia. In present study, we aimed to study the thyroid function tests of the anemic subjects and to compare characteristics and laboratory features of the three groups; hypothyroid, hyperthyroid, and euthyroid subjects. Methods: Anemic subjects divided into three groups according to the thyroid hormone status, either as hyperthyroid, hypothyroid and euthyroid groups. Hemogram indices and laboratory parameters compared between three groups. Results: Mean red cell distribution width (RDW) of hypothyroid anemic subjects was significantly lower than the RDW of euthyroid anemic subjects (p=0.003). White blood cell (WBC) count of hypothyroid anemic subjects was significantly reduced compared to the euthyroid (p<0.001) and hyperthyroid (p=0.047) anemic subjects. Significant inverse correlation between RDW and TSH (r=-0.25, p=0.001), between RDW and hemoglobin (r=-0.44, p<0.001), between RDW and hematocrit (r=-0.35, p<0.001) and between RDW and mean corpuscular volume (r=-0.53, p<0.001) were noted. Conclusions: Since anemia is common in thyroid conditions, besides its role in differential diagnosis of the anemia, RDW could also serve as an adjunct diagnostic tool in estimation of the thyroid hormone status in anemic subjects.

Edmonton frail score is associated with diabetic control in elderly type 2 diabetic subjects.

PURPOSE: We aimed to observe clinical and laboratory indices of the diabetic subjects who were either frail or not according to Edmonton frail score. We also aimed to study whether Edmonton frail score was correlated with metabolic and other parameters of the diabetic subjects. METHODS: Patients with T2DM visited our clinic were enrolled to the study and grouped as either frail or not frail according to the Edmonton score. Clinical and laboratory parameters of the groups were compared. RESULTS: Serum triglyceride (p = 0.04), serum albumin (p = 0.006) and Glomerular Filtration Rate (GFR) (p = 0.01) were significantly lower, while fasting blood glucose (FBG) (p = 0.02), HDL cholesterol (p = 0.005) and glycated hemoglobin (HbA1c) (p = 0.04) were significantly higher in frail group compared to the not frail patients. Edmonton frail score was positively correlated with HbA1c, age, duration of T2DM, FBG, and negatively correlated with serum albumin and GFR levels. CONCLUSIONS: We think that frailty is associated with poor glucose control in subjects with T2DM and better control of the disease may prevent or slow down the development of frailty, as well as microvascular complications in subjects with type 2 diabetes mellitus.

Is Neuregulin-4 a predictive marker of microvascular complications in type 2 diabetes mellitus?

BACKGROUND AND AIMS: Neuregulin-4 (Nrg-4) is a new adipokine released from brown adipose tissue. It plays pivotal role in regulating systemic energy balance, glucose and lipid metabolism and in reducing chronic inflammation. We aimed to investigate the relation between diabetic microvascular complications (DMC) and serum (Nrg-4) levels in patients with type 2 diabetes mellitus. METHODS: Patients with type 2 diabetes mellitus were divided into DMC and diabetic patients without microvascular complications (non-DMC). Nrg-4 levels of the patients were compared. RESULTS: Fifty and 29 patients enrolled to the DMC and non-DMC groups, respectively. Nrg-4 was 1.23 (0.02-5.1) ng/mL and 2.5 (0.21-6.01) ng/mL in DMC and in non-DMC groups, respectively (P < .001). In patients with DMC, FPG was 189.5 (89-446) mg/ dL, whereas it was 128 (95-278) mg/dL in non-DMC diabetic patients (P < .001). HbA1c was also significantly higher in the DMC group than in the non-DMC group (P < .001). Negative correlation was found between Nrg-4 and FPG (r = -0.231, P = .03), HBA1c (r = -0.312, P = .003) and microalbuminuria (r = -0.277, P = .009). Logistic regression analysis showed a 1-unit decrease in Nrg-4 to increase the presence of DMC by 1.9 times. The best cut-off value of Nrg-4 was 1.56 ng/mL with 82.1% sensitivity and 64% specificity, in predicting DMC. CONCLUSION: In patients with diabetes, Nrg-4 levels may be a good predictor of early detection of one or more DMC, as microvascular dysfunction in an organ system is considered to be an initial onset of subclinical systemic damage.

Association between novel inflammatory markers derived from hemogram indices and metabolic parameters in type 2 diabetic men.

AIM: Hemogram indices were proposed as novel inflammatory markers in chronic conditions and inflammation has substantial role in the pathogenesis of type 2 diabetes mellitus (T2DM). We aimed to observe hemogram parameters of type 2 diabetic male subjects with various age groups in well and poorly controlled subsets. METHODS: Data of type 2 diabetic men enrolled to the study were recorded from patient files of the institution. Study population were grouped into three according to the age. Group A was consisted of patients younger than 55 years, Group B was consisted of patients aged between 55 and 64 years, and Group C was consisted of patients aged 65 years or older. Data of the study groups were compared. RESULTS: Study population was consisted of 130 subjects; 44 in Group A and 43 in each of Groups B and C. Median red cell distribution width (RDW) (p = .04), mean RDW to platelet ratio (RPR) (p = .006), median mean platelet volume to platelet ratio (MPR) (p = .02) levels of the study groups were statistically different. HbA1c level was significantly and positively correlated with RDW (r = 0.45, p < .001), neutrophil to lymphocyte ratio (NLR) (r = 0.47, p < .001), mean platelet volume to lymphocyte ratio (MLR) (r = 0.35, p < .001), MPR (r = 0.26, p = .003), and RPR (r = 0.37, p < .001) levels. CONCLUSION: Elevated RDW, NLR, MLR, MPR, and RPR levels in diabetic men should trigger the measurement of HbA1c since each were strongly correlated with HbA1c level. Moreover, elevated RDW, NLR, MLR, and RPR could be marker of worse diabetic control in men with T2DM.

Type 2 diabetes mellitus is more commonly well controlled in younger men compared to older men.

METHODS: The male patients with T2DM that admitted to the outpatient internal medicine clinics of our institution between November 2018 and April 2019 were included to the study. According to the age, study population divided into two groups; either younger than 60 years (group I) and 60 years or older (group II). Study parameters were compared between study groups. RESULTS: Fasting plasma glucose (FPG) and glycated Hb level (HbA1c) of group I were lower than those of the group II (p = 0.004 for FPG and p = 0.048 for HbA1c). Control level of T2DM was defined as well controlled in 20 (29%) patients in group I and 5 (10.9%) patients in group II (p = 0.02). CONCLUSION: Physicians should aware that well controlled T2DM is more common among younger diabetic men compared to older men and try to enhance diabetic regulation level in elderly men by interventions that aimed to improve skeletal muscle or by pharmacologic agents.

Is Uric Acid elevation a random finding or a causative agent of diabetic nephropathy?

OBJECTIVE: In this study, we aimed to analyze the relationship between serum uric acid (UA) and microalbuminuria as a marker of renal injury in type 2 diabetes mellitus. METHODS: A total of 100 patients with type 2 diabetes mellitus were enrolled in the study. Participants were divided into two groups according to the urinary microalbumin/creatinine ratio: diabetic nephropathy and non-nephropathy group. UA and microalbuminuria were compared between the study groups. RESULTS: Serum UA levels of diabetic nephropathy patients were significantly higher than those in the non-nephropathy group (UA in patients with diabetic nephropathy groups: 6.3 (1.82) mg/dl, UA in patients of the non-nephropathic group: 4.85 (1.92) mg/dl) (p<0.001). There was a correlation between microalbuminuria and UA (r=0.238). This correlation was statistically significant (p=0.017). CONCLUSION: UA levels may be an important predictor of nephropathy in diabetic patients.

Neuregulin-4 is associated with plasma glucose and increased risk of type 2 diabetes mellitus.

BACKGROUND: Neuregulin-4 is a cytokine with many functions and is primarily produced by fat tissue. AIM OF THE STUDY: The aim of the study was to observe the relationship between serum neuregulin-4 levels and diabetes regulation in type 2 diabetes mellitus (T2DM), and to compare neuregulin-4 levels of diabetic subjects with those in healthy controls. METHODS: Patients with T2DM were included to the study. Healthy subjects were enrolled as controls. Subjects with T2DM with glycated haemoglobin (HbA1c) <7% were classed as well controlled and those with HbA1c >7% were classed as poorly controlled. Neuregulin-4 levels of the study and control groups were compared. RESULTS: The neuregulin-4 levels of the poorly controlled T2DM, well-controlled T2DM and control groups were significantly different (p = 0.005). Neuregulin-4 was significantly correlated with fasting plasma glucose (r = 0.247, p = 0.002) but not with HbA1c. In a regression analysis model, 0.1 point elevation in neuregulin-4 levels increased the rate of existence of T2DM 4.4-fold (odds ratio 4.4, 95% confidence interval 1.26-15.1; p = 0.02). CONCLUSION: Neuregulin-4 is significantly increased in patients with T2DM compared with control subjects, which means that it could be a marker of T2DM. Since neuregulin-4 was correlated with fasting glucose, we suggest that elevated neuregulin-4 could predict poor control in T2DM for short periods when HbA1c is not useful.  Moreover, one unit elevation in neuregulin-4 (0.1 ng/ml) increases the rate of existence of T2DM 4.4-fold, independently from other variables.

Is Uric Acid elevation a random finding or a causative agent of diabetic nephropathy?

SUMMARY OBJECTIVE In this study, we aimed to analyze the relationship between serum uric acid (UA) and microalbuminuria as a marker of renal injury in type 2 diabetes mellitus. METHODS A total of 100 patients with type 2 diabetes mellitus were enrolled in the study. Participants were divided into two groups according to the urinary microalbumin/creatinine ratio: diabetic nephropathy and non-nephropathy group. UA and microalbuminuria were compared between the study groups. RESULTS Serum UA levels of diabetic nephropathy patients were significantly higher than those in the non-nephropathy group (UA in patients with diabetic nephropathy groups: 6.3 (1.82) mg/dl, UA in patients of the non-nephropathic group: 4.85 (1.92) mg/dl) (p<0.001). There was a correlation between microalbuminuria and UA (r=0.238). This correlation was statistically significant (p=0.017). CONCLUSION UA levels may be an important predictor of nephropathy in diabetic patients.

RESUMO OBJETIVO O objetivo deste estudo foi analisar a relação entre o ácido úrico sérico e a microalbuminúria como marcador de lesão renal no diabetes mellitus tipo 2. MÉTODOS Um total de 100 pacientes com diabetes mellitus tipo 2 foram inscritos no estudo. Os grupos de estudo foram divididos em dois, de acordo com a relação microalbumina/creatinina na urina: nefropatia diabética e grupo não nefropático. UA e microalbuminúria foram comparados entre os grupos de estudo. RESULTADOS Os níveis séricos de AU de pacientes com nefropatia diabética foram significativamente maiores do que o grupo sem nefropatia (AU em pacientes com grupos de nefropatia diabética: 6,3 (1,82) mg/dl, AU em pacientes com grupos não nefropáticos: 4,85 (1,92) mg/dl ) (p<0,001). Houve correlação entre microalbuminúria e AU (r=0,238). Essa correlação foi estatisticamente significativa (p=0,017). CONCLUSÃO Os níveis de AU podem ser um importante preditor de nefropatia em pacientes diabéticos.

Neutrophil to lymphocyte ratio as an indicative of diabetic control level in type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus is associated with chronic low grade inflammation. One of the novel inflammatory markers is hemogram derived neutrophil to lymphocyte ratio (NLR). OBJECTIVE: We aimed to compare NLR levels of diabetic subjects and healthy controls and to observe possible correlation between NLR and HbA1c. METHODS: Medical data of type 2 diabetic subjects admitted to out-patient clinics of our institution between April to July in 2017 were obtained from database and retrospectively analyzed. Control group was chosen from healthy subjects who visited our institution for a routine check-up. Anthropometric measures, laboratory data, including, HbA1c, NLR were recorded. RESULTS: Median NLR of the type 2 DM group 2.44 (1.9) was significantly elevated when compared to healthy controls (1.5 (0.9), (p<0.001). In addition, a Pearson's correlation test revealed that NLR was strongly correlated with age (r=0.26, p=0.008), fasting plasma glucose (r=0.38, p<0.001), and HbA1c (r=0.49, p<0.001). CONCLUSION: Elevated NLR in otherwise healthy subjects may be indicative of underlying impaired glucose metabolism and moreover, NLR should be used as a marker of diabetic control level in addition to HbA1c in type 2 diabetic subjects.

Diabetic regulation of subjects with type 2 diabetes mellitus is associated with serum vitamin D levels.

OBJECTIVE: Vitamin D deficiency is not only associated with bone metabolism but also with diabetes mellitus. We aimed to study the possible association between serum vitamin D concentration and HbA1c level in patients with type 2 diabetes mellitus (T2DM) in this retrospective report. METHODS: Patients with T2DM were enrolled to the study either in regulated or non-regulated T2DM groups, according to HbA1c levels. An HbA1c level of <8% was considered as relatively controlled and others were considered as poorly controlled T2DM. RESULTS: Serum vitamin D levels in poorly controlled T2DM subjects (9.4 (4.9-34) ng/ml) were significantly lower than that of the relatively well regulated T2DM patients (13.5 (3.4-36) ng/ml) (p=0.03). Vitamin D was strongly and inversely correlated with HbA1c levels (r= -0.295, p=0.005). CONCLUSION: Whatever the cause or result of the diabetes mellitus, it is clear that lower vitamin D is strongly associated with worse diabetic regulation in T2DM subjects. Randomized controlled larger studies, which research the relation between diabetic regulation and vitamin D status, are needed to claim whether it could be a therapeutic target in future in diabetic subjects.

Diabetic regulation of subjects with type 2 diabetes mellitus is associated with serum vitamin D levels

SUMMARY OBJECTIVE Vitamin D deficiency is not only associated with bone metabolism but also with diabetes mellitus. We aimed to study the possible association between serum vitamin D concentration and HbA1c level in patients with type 2 diabetes mellitus (T2DM) in this retrospective report. METHODS Patients with T2DM were enrolled to the study either in regulated or non-regulated T2DM groups, according to HbA1c levels. An HbA1c level of <8% was considered as relatively controlled and others were considered as poorly controlled T2DM. RESULTS Serum vitamin D levels in poorly controlled T2DM subjects (9.4 (4.9-34) ng/ml) were significantly lower than that of the relatively well regulated T2DM patients (13.5 (3.4-36) ng/ml) (p=0.03). Vitamin D was strongly and inversely correlated with HbA1c levels (r= -0.295, p=0.005). CONCLUSION Whatever the cause or result of the diabetes mellitus, it is clear that lower vitamin D is strongly associated with worse diabetic regulation in T2DM subjects. Randomized controlled larger studies, which research the relation between diabetic regulation and vitamin D status, are needed to claim whether it could be a therapeutic target in future in diabetic subjects.

RESUMO CONTEXTO E OBJETIVO A deficiência de vitamina D não é apenas associada ao metabolismo ósseo, mas também ao diabetes mellitus. Procurou-se estudar a possível associação entre os níveis de concentração do soro de vitamina D e de HbA1c em pacientes com diabetes mellitus tipo 2 neste relatório retrospectivo. MÉTODOS Os pacientes com diabetes mellitus tipo 2 foram inscritos no estudo em regulada ou não regulada de acordo com os grupos de níveis de HbA1c DM2. HbA1c nível de <8% caracterizava DM2 controlada e HbA1c > 8% DM2 descontrolada. RESULTADOS Os níveis de vitamina D no soro em indivíduos com DM2 mal regulados (9,4 (4,9 a 34) ng/ml) foram significativamente menores do que o do bem regulado em doentes DM2 (13,5 (3,4-36) ng/ml) (p = 0,03). A vitamina D foi forte e inversamente correlacionada com os níveis de HbA1c (p = 0,005). CONCLUSÃO Seja qual for a causa ou o resultado do diabetes mellitus, é claro que níveis baixos de vitamina D são fortemente associados com pior regulação em indivíduos diabéticos com DM2. Maiores estudos randomizados e controlados que pesquisam a relação entre o status de vitamina D e a regulação em diabéticos são necessários para molusco se é, no futuro, poderia ser um alvo terapêutico em indivíduos diabéticos.

Mean Platelet Volume to Lymphocyte Ratio as a Novel Marker for Diabetic Nephropathy.

OBJECTIVE: To compare mean platelet volume to lymphocyte ratio (MPVLR) of diabetic nephropathy subjects to those diabetics without diabetic nephropathy. STUDY DESIGN: Observational, cross-sectional study. PLACE AND DURATION OF STUDY: Tertiary referral hospital, in Bolu, Turkey, from July to December 2017. METHODOLOGY: Patients with type 2 diabetes mellitus admitted to the Internal Medicine Clinic, were included. Patients were divided into two groups according to the urinary microalbumin/creatinine ratio: diabetic nephropathy and non-nephropathy group. MPVLR was calculated mathematically by division of MPV by lymphocyte count. RESULTS: There were 162 subjects (79 females, 83 males). The MPVLR in patients with diabetic nephropathy and non- nephropathic diabetic groups were 4.1 (2.09-11.84) and 3.4 (1.37-25.56), respectively. The difference was reached statistically significant level (p <0.001). The best cut-off value for MPVLR was 3.66 (AUC=0.733, p <0.001); MPVLR predicted diabetic nephropathy with 71.1% sensitivity and 67.4% specificity, at this level. CONCLUSION: The MPVLR is an easily calculated and efficient index that can be considered a powerful and independent predictor of diabetic nephropathy in diabetic patients. We suggest that, it can be useful adjunct to standard tests in the diagnosis of diabetic nephropathy.

A Rare Non-Hemolytic Case of Idiopathic Cold Agglutinin Disease.

BACKGROUND: Cold agglutinin disease is a very rare condition associated with agglutination of erythrocytes in cold environment usually due to IgM type antibodies. Other than hemolytic anemias, it may interfere with routine hemogram tests due to miscalculation of red blood cell count (RBC) and other hemogram parameters calculated with involvement of RBC. Awareness of the condition is important to overcome laboratory errors. METHODS: We studied a peripheral blood smear and repeated the hemogram test at 37°C to establish the diagnosis of cold agglutinin disease. RESULTS: Initial hemogram test results of the fifty-eight year-old man was as follows: RBC: 1.34 M/µL, hemoglobin (Hb): 12.4 g/dL, hematocrit (Htc): 11.8%, mean corpuscular hemoglobin (MCH): 92.4 pg, and mean corpuscular hemoglobin concentration (MCHC): 105 gr/dL. Despite the standard indirect Coombs test being negative, repeated tests at room temperature was 4+. We suspected cold agglutinin disease and repeated the hemogram test using the Bain-Marie method at 37°C and the test results showed RBC: 3.4 M/µL, hemoglobin: 12.6 g/dL, hematocrit: 30.2%, MCH: 31.7 pg, and MCHC: 41.8 g/dL. CONCLUSIONS: Inappropriate hemogram results may be a sign of underlying cold agglutinin disease. Hemolytic anemia not always accompanies the disease; however, cold exposure may trigger erythrocyte agglutination in vitro and may cause erratic laboratory results.

In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells.

AIMS/HYPOTHESIS: We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. METHODS: Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice. RESULTS: The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols. CONCLUSIONS/INTERPRETATION: These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.

Phenotypic consequences of lung-specific inducible expression of FGF-3.

Members of the fibroblast growth factor (FGF) family play a critical role in embryonic lung development and adult lung physiology. The in vivo investigation of the role FGFs play in the adult lung has been hampered because the constitutive pulmonary expression of these factors often has deleterious effects and frequently results in neonatal lethality. To circumvent these shortcomings, we expressed FGF-3 in the lungs under the control of the progesterone antagonist-responsive binary transgenic system. Four binary transgenic lines were obtained that showed ligand-dependent induction of FGF-3 with induced levels of FGF-3 expression dependent on the levels of expression of the GLp65 regulator as well as the dose of the progesterone antagonist, RU486, administered. FGF-3 expression in the adult mouse lung resulted in two phenotypes depending on the levels of induction of FGF-3. Low levels of FGF-3 expression resulted in massive free alveolar macrophage infiltration. High levels of FGF-3 expression resulted in diffuse alveolar type II cell hyperplasia. Both phenotypes were reversible after the withdrawal of RU486. This system will be a valuable means of investigating the diverse roles of FGFs in the adult lung.

Transgenic mouse models for lung cancer.

Transgenic technology allows the ability to target regulatory genes to the lungs in a cell-specific fashion. Using this technology, we have generated a model to investigate the phenotypic consequences of targeting oncogenes to particular cell types in the lungs and are developing a second model for the regulated expression of oncogenes in the lung. The transgenic model involves the constitutive expression of simian virus 40 large T antigen in the Clara cells of mouse lungs. This model has been used to investigate changes in expression of cell cycle regulatory genes in the Clara cells during the transformation process, as well as the expression of the transcription factors regulating the expression of Clara cell differentiation markers. The second model we are developing investigates the regulated expression of the genes in the lungs. This system is based on the establishment of two types of transgenic lines. The regulator line consists of a chimeric transcriptional factor placed under the control of a lung-specific SPC (surfactant protein C) promoter. This chimeric regulator is composed of a transcription activation domain, the GAL4 DNA-binding domain, and a truncated progesterone receptor that is responsive to RU 486, but not to endogenous progesterone. The second transgenic mouse line contains the silent target transgene under the control of a minimal promoter with upstream activating sequences (UAS) that are recognized by the regulator transgene. Upon breeding these two lines, the resulting bitransgenic mice can then be induced to express the target transgene only with the administration of RU 486. Two generations of regulators have been evaluated on their ability to regulate the expression of a growth hormone reporter gene. This system demonstrated the inducible expression of the reporter genes in the distal airways of the lungs.

Ligand-dependent regulation of plasmid-based transgene expression in vivo.

As gene therapy advances, the ability to regulate transgene expression will become paramount for safety and efficacy. In this study, we investigate the ability of the mifepristone-dependent GeneSwitch system to regulate the expression of trangenes delivered to mice by nonviral methods. Two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene for secreted human placental alkaline phosphatase (SEAP), were delivered to the hind-limb muscles of adult mice. Modulation of the level of secretion of the transgene product into serum was achieved by intraperitoneal administration of low doses of the drug mifepristone (MFP). The EC50 for induction of transgene expression by MFP was 0.03 +/- 0.005 mg/kg. The maximal level of transgene expression after induction was equal to or higher than that displayed by a plasmid driven by the CMV enhancer/promoter. The average magnitude of induction was 14- to 19-fold. Multiple rounds of drug-dependent regulation of transgene expression in vivo were demonstrated. In BALB/c mice, the ability to regulate transgene expression persisted for approximately 3 weeks, until the appearance of neutralizing antibodies to the secreted transgene product. In immune-deficient mice, the ability to repetitively regulate transgene expression persisted for at least 5 weeks. Although the dynamic range of regulation needs improvement, the plasmid-based GeneSwitch system has features that are attractive for gene therapy applications.

Adenovirus-mediated regulable target gene expression in vivo.

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor-ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-kappaB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.