Revista Facultad de Salud - RFS Enero - Junio 2010 • Universidad Surcolomt)íana • Neiva - Huila Vol. 2 Nra. 1 - 2010: 21-29

ARTÍCULO DE INVESTIGACIÓN

Doris M. Salgado¹, Marisol Garzón*, Rocío Vega*, Cesar Panqueba*,

Carlos F. Narváez*, Jairo A. Rodríguez

PATTERN OF SERUM CYTOKINES IN CHILDREN WITH DENGUE HEMORRHAGIC FEVER IN NEIVA, COLOMBIA

Patrón de citoquinas en niños con dengue hemorrágico en Neiva, Colombia

Fecha de recibido: 11 de abril de 2010 • Fecha de aprobación: 30 de mayo de 2010

Abstract. Cytakines play a critical rale in the patha-genesis af dengue hemarrhagic fever and have been used as markers af severity disease. In this study we measured serum levels af five cytakines in dengue infected children and carrelate their levels with shack and camplicated farms such as myacarditis, hepatitis ar bleeding.

Methods: 30 patients wha met WHO criteria far dengue hemarrhagic fever (DHF) were enralled and classified inta twa graups: graup ane withaut shack (grade I and II) and graup twa with shack (grade III and IV). Serum levels af TNFa, IL-6, IL-10, IL-4 and IFNy were measured by ELISA at first day af defervescence and were campared with serum levels af 28 healthy children.

Results: fram 30 patients, 9 were assigned ta graup number ane (median age 67 manths) and 21 ta graup twa (median age 42 manths). Statistical differences were faund between dengue infected patients and cantrals: cantrals IL-6 (5.2 pg/ml), graup 1 (485 pg/ml) (p=0.002) and graup 2 (190 pg/ml) (p=0.001); TNFa, cantral graup (70 pg/ml), graup 1 (586.7 pg/ml) (p=0.021) and graup 2 (320.7 pg/ml) (p<0.001) and far IFNy, cantral graup (12.3 pg/ml), graup 2 (27.5 pg/ml) (p=0.019). Hawever, we cauld nat find carrelatian between cytakines and shack ar camplicated farms af illness. IL-4 and IL-10 did nat shaw differences between any tested graups.

Conclusion: IL-6, TNFa and IFNy are elevated in children with dengue hemarrhagic fever, but there was nat carrelatian with severe farms af shack.

Key words: Dengue, Dengue hemarrhagic fever (DHF), shack dengue syndrame (SDS), Cytakines.

Resumen. Las citaquinas juegan un papel crítica en la patagénesis de la fiebre dengue hemarrágica (FDH) y han sida usadas cama marcadares de severidad. En este estudia se midieran las niveles de cinca citaquinas en niñas infectadas can dengue y se carrelacianaran can el chaque y las farmas camplicadas tales cama mia-carditis, hepatitis a sangrada.

Método: 30 pacientes que cumplían las criterias de la OMS para FDH fueran incluidas y clasificadas en das

grupas: grupa 1, sin choque (grada I y II) y grupa 2, can chaque (grada III y IV). Niveles séricas de TNFa, IL-6, IL-10. IL-4 e IFN y fueran medidas par ELISA en el primer día de la defervescencia y comparadas can las respectivas niveles de 28 niñas sanas.

Resultados: de las 30 pacientes, 9 fueran clasificadas en el grupa número 1 (media de edad de 67 meses) y 21 en el grupa 2 (media de la edad 42 meses). Diferencias estadísticamente significativas fueran encantradas entre niñas infectadas can dengue y cantrales sanas: sanos IL-6 (5,2 pg/ml), grupa 1 (485 pg/ml) (p = 0,002) y grupa 2 (190 pg/ml) (p = 0,001); TNFa, grupo control (70 pg/ml), grupo 1 (586,7 pg/ml) (p = 0,021) y grupo 2 (320,7 pg/ml) (p < 0,001) y para IFNy, grupa cantral (12,3 pg/ml), grupa 2 (27,5 pg/ml) (p = 0,019). Sin embarga, na se encontró correlación entre las citaquinas y el choque a las otras formas evaluadas. IL-4 e IL-10 na fueran diferentes en ninguna de las grupas analizadas.

Oanclusión: IL-6, TNFa e IFNy están elevadas en niñas can FDH, pera na huba carrelación can las farmas severas de chaque.

Palabras clave: dengue, fiebre dengue hemarrágica (DH), síndrame de chaque par dengue (SSD), citaquinas.

INTRODUCTION

Dengue hemarrhagic fever (DHF) has emerged as the mast impartant arbavirus disease in man in the last three decades. It has been estimated that about 50 million cases of DF occur every year with about 250,000 to 500,000 cases of DHF⁽¹⁾. In 2001 there was an unprecedented gla-bal dengue epidemic activity in American cauntries, the Pacific islands and Asia. During 2002, mare than 30 Latin American cauntries reported over 10,000,000 DF cases with large number of DHF cases⁽²⁾. In Oolombia global incidence rate is about 13 cases for 100,000 habitants but in same regians such as Santander or Huila this rate is higher than 60 cases for 100,000 and has become a leading cause of haspitalizatian and death amang children⁽³⁾.

Dengue virus (DV) infectian has a spectral presentatian fram asymptamatic farms ta dengue fever (DF) and dengue hemarrhagic fever (DHF) with life threatening situatians af shack, bleeding or more recently described complicated atypical forms like hepatitis, encephalitis, myacarditis^(4-6).

Studies have shawn the cytakines as a pivatal piece in the pathagenesis af dengue hemarrhagic fever that reflects an imbalance between T helper

1 (Th1) and Th2 response. Thus, an early Th1 response has been characterized with a production of IFNy and IL-2 which mediate cellular activatian needed far viral clearance and later a Th2 respanse with IL-4, IL-5, IL-6, IL-10 and IL-13 release which are responsible for pro inflammatory effect with endothelium and hameastatic alteratians leading ta shack and bleeding forms⁽⁷⁾. Recently, others cytokines such as IL8(⁸), IL-1Ra(⁹), IL-12(i°), hCTF(⁷), MIF(H) has been obtained from another cells such as endathelium, neutraphil, dendritic cells and hepatocytes when these are infected by dengue virus, which gives us an idea of complexity of cytakine rale in dengue hemarrhagic fever. Hawever, exist a variatian af cytakines pattern accarding ta dengue virus seratype and HLA palymarphism⁽¹²⁾ that cauld explain individual patterns of cytokine in each population and why same regians have mare tendency ta dengue hemarrhagic fever with severe and camplicated farms. In this study we will determine cytakines prafile in a papulatian af children fram Neiva, Oolombia and to correlate with severity of disease.

MATERIALS AND METHODS Patient enrollment

This study included thirty patients under 13 years old who were admitted to Pediatric Department of Hospital Universitario de Neiva between june 2004 ta June 2005. Entry criteria were clinical diagnasis af DHF using WHO criteria before the results of serological studies were knawn and severity af illness was graded according to the presence of shock into two graups: graup ane withaut shack and graup two with shock. 28 healthy children in the out patient department served as a cantral graup. Epidemialagical and clinical dates were recarded at time af day sixth when measure af cytakines were made.

The study was approved by the Medical ethics Committee of Medicine program from Universidad Surcolombiana and by the ethics committee af the Haspital Universitaria de Neiva. Infarmed consent was obtained from the parent of each patient included in the study.

Laboratory testing

Blood sample was obtained at first day of defervescence (+2 day of disease). Oytokines TNFa, IL-6, IL-10, IL-4 and IFNy were measured with an ELISA technique (Anogen, Ontario, CA) according to the manufacturer's instructions. These cytokines were measured also in healthy patients as control. The limit detection of every cytokine was: IL-4 (4.3pg/mL), IL-6 (1.7pg/mL), IL-10 (2pg/mL), TNFa (4pg/mL) and IFNy (7.5pg/mL). Standards were included in each assay and the curve was used for estimation of cytokine concentration (in pg/mL) by regression analysis. Ig M for dengue virus was made using ELISA technique as well (PanBio).

Coagulation test (prothrombine time, partial thromboplastine time and platelets count), liver function test (alanine aminotransferase-ALT, aspartate aminotransferase-AST) and electro-cardiograph were made to determine liver, haemostatic and myocardium involvement respectively.

Date analysis

All data are presented as median and range. Differences in levels of IL-6, TNFa, IL-10, IFNy and IL-4 between cases and controls as well as between controls, shock and no shock patients were analyzed by the Kruskal-Wallis follow by Dunn's multiple comparison test. P value of less than 0.05 was considered to represent a signifi-cant difference. All statistical calculations were done using SPSS 6.0 and Graphpad prism 4.0 software.

RESULTS

From a total of thirty patients, nine (30%) were classified in the group without shock and twenty one (70%) in the group with shock from whom, five patients had profound shock with narrow of pulse pressure lesser than 20 mmHg.

The median age, gender distribution and signs and symptoms in each group are presented in Table 1. Forty three percent of patients included were younger than 4 years old with a youngest has 6 months and older has 12 years. We observed that patients in the group with shock were younger (median age 48 months) and showed more symptoms compared to group without shock (median age 74 months).

Patients has a median of fever 4.6 days because we are reference hospital. Between clinical signs hepatomegaly was a constant finding followed by pleural effusion (median 30%) and bleeding in a 60% of patients in both groups.

Between laboratory test, platelets count were under 50,000x mm³ in around 50% of patients and less of 20,000x mm³ in 20% of patients; prothrombine time has a median of 20 sec with a higher value of 118" and partial thromboplastine time has a mean value of 65" with a higher value of 124".

Aminotransferase levels were higher for AST with a median value of 403.1 U/l in both groups (227 U/l group 1 and 175 U/l group 2) compared to ALT levels of 171.5 U/l (81 u/l group 1 and 127 U/l group 2) with AST/ALT ratio of 2.3 as were described for hepatitis dengue.

Electrocardiograph was abnormal in 8 patients with rhythm alteration and ST changes however echocardiogram was not available in these patients.

Despite clinical and laboratory test were more abnormal in patients with shock we did not find statistically differences between groups for any of this variables.

Figure 1. Pattern of serum levels of cytokines in healthy controls, dengue infected children without or with shock. Levels of cytokines were detected by ELISA six days after fever beginning. Lines represented the median. Kruskal-Wallis P values are shown.

Based in clinical and laboratory findings were identified: 11 (36.6%) cases of hepatitis, 8 (26.6%) cases suggestive of myocarditis and 13 (43.3%) cases of bleeding.

Pattern of cytokines in dengue infected children

Serum levels of TNFa, IL-6, IFNy, IL-10 and IL-4 in patients with dengue hemorrhagic fever in all groups are summarized in Figure 1. P of Kruskal-Wallis test were < 0.01 in all the cytokines analyzed. We found statistically differences for IL-6 between controls and group one (p = 0.001) and group two (p = 0.001); for TNFa between controls and group one (p = 0.001) and group two (p< 0.001) and IFNy between controls and shock patients (p = 0.019). Of note, serum levels of TNFa and IL-6 were higher in patients without shock but there were not statistically difference between shock and no shock groups.

IL-4 and IL-10 did not show statistical differences between healthy and dengue infected patients (Figure 1).

Figure 2. Hepatitis and bleeding are not related with TNFa or IL-6 levels. (A). No correlation was found between TNFa or IL-6 with AST (marker of hepatic injury) values (A) or bleeding sings (PTT and platelet number) (B).

Na carrelatian between levels af TNFa ar IL-6 with a marker af Hepatic injury (AST) was faund (Figure 2A). On the other hand, bleeding paraclinical signs (platelet number and PTT) were nat clearly assaciated with and IL-6, TNFa and IL-10 levels as well.

Median days af stay was 6 and there were nat deaths.

DISCUSSION

DF and DHF is an endemic-epidemic disease in Huila. Althaugh there is a shift taward yaung adults⁽¹⁾ severe forms of DHF with shock cantinue ta be mare frequent in children as is shawed in this study almast under five years ald; symptams such as vamiting, bleeding, abdaminal pain are still alarm signs in dengue hemarrhagic fever because they are carrelated with plasma leakage and shack.

In this study there was a higher incidence af hepatitis, myacarditis and bleeding in patients with shack thus, these camplicated farms can be cansidered such a markers af severity. The liver invalvement has been studied fram asian epidemics where patients shawed necrasis and hepatitis in facal and generalized farms with apaptatic changes similar ta findings in yellaw fever. There after, studies shawed ta hepatacytes and Kuppfer cells such as target of the virus and contribute to cytokines and mediators release such as IL-6, O-reactive protein and secretory phasphalipase A2 (sPLA2) type II, which in turn may warse inflammatary respanse⁽¹³⁾ Olinical studies have carrelated severity with amina-transferase levels, an increase more than 10 folds narmal value are cansidered such a marker af severity. It is cansistent with aur results that shaw high levels af aminatransferase specifically aspartate aminatransferase levels (AST, mean 403 U/l).

Bleeding signs were present in 43% of patients with alteratian in caagulatian test. There are many explanatians ta haemastatic alteratians in DHF /DSS such as a law praductian af caagula-tion factors due to liver involvement, imbalance between caagulatian and fibrinalysis and an immune respanse against platelets and enda-thelium which are carrelated ta disease severity.

Myacarditis an unusual camplicatian has been described as a mild bradycardia in late phase with few clinical signs and edema and hema-rrhage in autopsies⁽¹⁴⁾. However, in the last two years a severe farm af myacarditis assaciated ta dengue virus has been reparted in aur regian with a fatality case af fulminant myacarditis^{(15, 16)}. In this study a 23% af patient had rhythm changes suggestive af myacarditis hawever it lacks af anather test ta canfirm this diagnasis. Ta extent the analysis af the myacarditis, we recently shaw at the first time, the direct infectian af human muscle cardiac cells by dengue virus in vivo⁽¹⁷⁾.

It has been suggested that cytokines and chemical mediatars play a rale in the patha-genesis of DHF /DSS. One of the cells res-pansible af cytakine praductian is T lymphacyte that releases several cytokines including IFNy, IL-2, IL-4, IL-5, IL-6, IL-10, and lymphotoxin. Monocytes/macrophages also produce TNFa, IL-1, IL-1b, IL-6, and PAF⁽¹⁸⁾, furthermore, some reparts have demanstrated the praductian af cytakines ar chemakines after in vitro DV infection of different types of cells, including endathelial cells^(19,20), blaad manacytes⁽²¹⁾, liver Kuppfer cells⁽²²⁾, mast cell/basophil line⁽²³,²⁴⁾ thus, cytakine respanses is prapartianal ta the infected cell mass⁽²⁵⁾.

The studies cytakines functians in dengue infectian came fram in vitro assays with specific cell bulk. In the clinical context it results difficult due to the complexity of cytokines network because they can activate cells synergistically ar antaganistically and the net autcame will depend an the balance between variaus cytakine actians. It is clear that each cytokine pattern belong to a specific T helper respanse that will depend an antigen challenge. In dengue virus, studies show a Th1 pattern (IFNy -IL2) in early phase of diseases and shift taward a Th2 respanse (IL4, IL5, IL6, IL10, IL13) in the late phase⁽²⁶⁾. From culture supernatants on infected cells, cytokines recavered an the first day past-infectian were human cytotoxic factor (hOF), TNFa, IL-2 and IL-6; their levels reached a peak on the second day, IFNy appeared on the 2nd day with a peak on the 3rd day, IL 10, IL5 start later on the 4th day and IL-4 on the 6th day⁽²⁷⁾.

Nguyen et al. showed that infants with DHF/ DSS had levels of IFNy in the acute-phase significantly higher than those in samples from healthy control (56.2 vs. 4.1 pg/ml) and were increased on day 4 to day 6 after the onset of fever and rapidly decreased on day 7 and during the convalescent phase, similar results were observed with TNFa levels which were elevated on day 4 to day 7 after the onset of fever and decreased on day 8 to day 19; IL-10 and IL-6 levels were also detected in the acute-phase but a lesser degree, thus is observed a Th1 predominan! response in acute phase⁽²⁵⁾. Kurane et al., described T cell response in children with DHF/ DSS; they found IL-2 levels > 10 U/ml in 63% (26/41) of patients during days 3-8 after onset of fever and IFNy was detected in 97% (34/35) of the patients with DHF on days 3-8 (p < 0.001 on days 3-6 and p < 0.02 on days 7-8) Our results presented in fig 1, shown a significant elevated levels of serum IFNy in shock group. This cytokine is specially produced by T cells and it will be consistent and indicate that T lym-phocytes are activated in vivo during DHF⁽²⁸⁾.

In our study, cytokines measured at sixth day showed important levels of IFNy, TNFa and IL6 in DHF compared to controls but IL10 and IL4 levels were similar to healthy patients, which reflect a predominant and persistent Th1 response in our patients. We could not find correlation with severity and higher levels of TNFa and IL-6 were seen in patients without shock. In this regard, other studies have found similar results. Kurane in the study mentioned above, did not find significant differences in the levels of sIL-2R, sCD4, sCD8, IL-2, and IFNy among grades I, II, and III of DHF. Analysis in the Nguyen's study, do not found statistical differences in serum levels of IFNy, TNFa, IL-10, and IL-6 between patients with no shock DHF and those with DSS, but a significantly higher elevation of IL-6 was observed in patients who died than in patients who survived the infection.

Despite of these clinical correlation were not conclusive, there are many studies which shown individual correlation of cytokine and severity. Elevated serum levels of TNFa and IL-6 as well as their association with severity of the disease and with DHF/DSS have been reported in human dengue infection⁽²⁹⁾. One fatal case in Rio de Janeiro, Brazil is described in one patient with serum level of 900 pg/ml TNFa⁽³⁰⁾. Experimental studies with anti TNFa based on the pre-ter-minal TNFa peak seen in lethal dengue model, found that animals treated with anti-TNFa serum have a better survive⁽³¹⁾. Patients from the study showed very high serum levels of TNFa in dengue hemorrhagic patient compared to healthy patients. This finding can explain the high proportion of DHF vs DF seen in children from our region due to the important role of TNFa in endothelium activation leading to plasma leakage and bleeding forms.

The IL-6 plays also a central role in pathogenesis of dengue due to its wide range of immune and hematopoietic activities and also its potential ability to induce the acute phase response, induction of terminal differentiation of B cells and activation of T cells. Different studies des-cribed IL-6 increased after dengue virus infection. Some studies show a correlation between severity of illness and IL-6 levels. One study in 186 children showed IL6 levels correlated with shock in DHF, they found higher plasma levels of IL-6 in the shock group than in no shock group (p = 0.05) however, although IL-6 levels were somewhat higher in the DHF patients than in the DF patients, this did not reach statistical significance. Another study in 24 patients with DHF (4 to 75 years old) showed levels of IL-6 significantly elevated in DHF/DSS patients but not in DF patients, compared to normal controls or patients with other febrile illness⁽³²⁾. In contrast with these studies, we found higher levels in DHF patients than controls but this cytokine was higher in the group without shock similar to a prospective study⁽²⁵⁾, in which IL-6 levels tended to be lower in patients in shock than those not in shock. This finding could be explaining by the cytokines kinetic where the highest levels of IL-6 are seen around 3th day and decline thereafter. In this study patients were enrolled around sixth day when the clearance of this cytokine has begin.

IFNy could have a double function, one is the viral clearance thanks to T cells and DC activation, and by the other hand could facilitate the antibody-depending enhancement (ADE) due to a up-regulate of the expression of FCy receptors. The increased number of dengue vi-rus-infected monocytes/macrophages results in an elevation of T-cell activation, which results in the release of high levels of cytokines and chemical mediators with an increase in vascular permeability, plasma leakage, shock, and malfunction of the coagulation system⁽¹⁸,³³⁾ which coincide with our finding of higher levels in the group with shock (Figure 1).

It has been show that IFNy was able to decrease the infection and virus titer in dendritic cells at 48 h⁽¹⁰⁾. This is supported by clinical studies which show an elevation of IFNy in early phase on or before the day of defervescence, coinciding with disappearance of viraemia⁽³⁴⁾.

When serum levels of cytokines measured in children from the study are compared to other endemic regions for dengue we found a very high levels of TNFa and IL-6 (Figure 1). This fact can be explaining by an individual variation in HLA context⁽²⁵,³⁵⁾ that could be contributing to a more severity, shock and complicated forms seen in our patients. Consistent with that interpre-tation, a recent study in two ethnic colombian groups (afro-colombian and mestizos) show important and significant differences between the cytokine pattern present in patients with dengue fiver and DHF⁽³⁶⁾.

However we could not demonstrated correla-tion between cytokines and shock, myocarditis, hepatitis and bleeding forms probably to be measured at specific point of time (sixth day) where the kinetic behavior of cytokines could not be seen.

In conclusion, at sixth day of disease our patients show a still a high proinflammatory and Th1 response which contribute to understanding of the disease and can support the hypothesis of benefit with immunomodulatory therapy in these patients.

ACKNOWLEDGMENTS

We would like to thank children enrolled in the study and the Medical and Paramedical personal of the Hospital Universitario de Neiva and Yamileth Monje for technical assistance. This study was supported by a grant from Vicerrec-toría de Investigación, Universidad Surcolom-biana, Neiva, Colombia.

REFERENCES

1. World Health Organization. Dengue guidelines for diagnosis, treatment, prevention and control: new edition. Geneva: World Health Organization; 2009.

2.    Chaturvedi UC, Shrivastava R, Nagar R. Dengue vaccines: problems and prospects. ludían J Med Res 2005;121(5):639-652.

3.    Instituto Nacional de Salud. Análisis epidemiológico semana 35. Boletín Epidemiológico Instituto Nacional de Salud. 2006.

4.    Nimmannitya S, Thisyakorn U, Hemsrichart V. Dengue haemorrhagic fever with unusual manifestations. Southeast Asian J Trop Med Public Health 1987;18(3):398-406.

5.    Solomon T, Dung NM, Vaughn DW, Kneen R, Thao LT, Raengsakulrach B, et al. Neurological manifestations of dengue infection. Lancet 2000;355(9209):1053-1059.

6.    Kamath SR, Ranjit S. Clinical features, complications and atypical manifestations of children with severe forms of dengue hemorrhagic fever in South India. Indian J Pediatr 2006;73(10):889-895.

7.    Chaturvedi UC. The curse of dengue. Indian J Med Res 2006;124(5):467-470.

8.    Bosch I, Xhaja K, Estevez L, Raines G, Melichar H, Warke RV, et al. Increased production of interleukin-8 in primary human monocytes and in human epithelial and endothelial cell lines after dengue virus challenge. J Virol 2002;76(11):5588-5597.

9.    Suharti C, van Gorp EC, Dolmans WM, Setiati TE, Hack CE, Djokomoeljanto R, et al. Cytokine patterns during dengue shock syndrome. Eur Cytokine Netw 2003;14(3):172-177.

10.    Libraty DH, Pichyangkul S, Ajariyakhajorn C, Endy TP, Ennis FA. Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis. J Virol 2001;75(8):3501-3508.

11.    Chen LC, Lei HY, Liu CC, Shiesh SC, Chen SH, Liu HS, et al. Correlation of serum levels of macrophage migration inhibitory factor with disease severity and clinical outcome in dengue patients. Am J Trop Med Hyg 2006;74(1):142-147.

12.    Stephens HA, Klaythong R, Sirikong M, Vaughn DW, Green S, Kalayanarooj S, et al. HLA-A and -B allele associations with secon-dary dengue virus infections correlate with disease severity and the infecting viral serotype in ethnic Thais. Tissue Antigens 2002; 60(4):309-318.

13.    Juffrie M, van Der Meer GM, Hack CE, Haasnoot K, Sutaryo, Veerman AJ, et al.

Inflammatory mediators in dengue virus infection in children: interleukin-8 and its relationship to neutrophil degranulation. Infect Immun 2000;68(2):702-707.

14.    Promphan W, Sopontammarak S, Pruekprasert P, Kajornwattanakul W, Kongpattanayothin A. Dengue myocarditis. Southeast Asian J Trop Med Public Health 2004;35(3):611-613.

15.    Salgado D, Panqueba C, Vega R, Garzón M, Castro D, Rodríguez, J. Mortalidad por dengue hemorrágico en niños en Colombia: más allá del choque. Infectio 2008;12(1):247-253.

16.    Salgado DM, Panqueba CA, Castro D, M RV, Rodriguez JA. [Myocarditis in children affected by dengue hemorrhagic fever in a teaching hospital in Colombia]. Rev Salud Pública (Bogotáa). 2009;11(4):591-600.

17.    Salgado DM, Eltit JM, Mansfield K, Panqueba C, Castro D, Vega MR, et al. Heart and Skeletal Muscle Are Targets of Dengue Virus Infection. Pediatr Infect Dis J 2010 Dec 21.

18.    Green S, Rothman A. Immunopathological mechanisms in dengue and dengue hemorr-hagic fever. Curr Opin Infect Dis 2006;19(5):429-436.

19.    Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M. Dengue virus infection of human endothelial cells leads to chemokine produc-tion, complement activation, and apoptosis. J Immunol 1998 Dec. 1;161(11):6338-6646.

20.    Warke RV, Xhaja K, Martin KJ, Fournier MF, Shaw SK, Brizuela N, et al. Dengue virus induces novel changes in gene expression of human umbilical vein endothelial cells. J Virol 2003;77(21):11822-11832.

21.    Kurane I. Dengue hemorrhagic fever with special emphasis on immunopathogenesis. Comp Immunol Microbiol Infect Dis 2007;30(5-6):329-340.

22.    Marianneau P, Steffan AM, Royer C, Drouet MT, Jaeck D, Kirn A, et al. Infection of primary cultures of human Kupffer cells by Dengue virus: no viral progeny synthesis, but cytokine production is evident. J Virol 1999;73(6):5201-5206.

23.    King CA, Anderson R, Marshall JS. Dengue virus selectively induces human mast cell chemokine production. J Virol 2002;76(16):8408-8419.

24.    King CA, Marshall JS, Alshurafa H, Anderson R. Release of vasoactive cytokines by antibody-enhanced dengue virus infection of a human mast cell/basaphil line. J Virol 2000;74(15):7146-7150.

25.    Nguyen TH, Lei HY, Nguyen TL, Lin YS, Huang KJ, Le BL, et al. Dengue hemarrhagic fever in infants: a study af clinical and cytakine prafiles. J Infect Dis 2004;189(2):221-232.

26.    Ohaturvedi UO. Shift ta Th2 cytakine respanse in dengue haemarrhagic fever. Indian J Med Res 2009;129(1):1-3.

27.    Ohaturvedi UO, Elbishbishi EA, Agarwal R, Raghupathy R, Nagar R, Tandan R, et al. Sequential praductian af cytakines by dengue virus-infected human peripheral blaad leukacyte cultures. J Med Virol 1999;59(3):335-340.

28.    Kurane I, Innis BL, Nimmannitya S, Nisalak A, Meager A, Janus J, et al. Activatian af T lym-phacytes in dengue virus infectians. High levels af soluble interleukin 2 receptar, soluble OD4, soluble OD8, interleukin 2, and interferan-gamma in sera af children with dengue. J Clin Invest 1991;88(5):1473-1480.

29.    Azereda EL, Zagne SM, Santiago MA, Gauvea AS, Santana AA, Neves-Sauza PO, et al. Oha-racterisatian af lymphacyte respanse and cytakine patterns in patients with dengue fever. Immunobiology 2001;204(4):494-507.

30.    Kubelka OF, Barges PA, VanSydaw FF, Lampe E. Analysis af tumar necrosis factar-alpha serum level in Brazilian patients with

Dengue-2. Mem Inst Oswaldo Cruz 1995;90(6):741-742.

31.    Atrasheuskaya A, Petzelbauer P, Fredeking TM, Ignatyev G. Anti-TNF antibady treatment reduces martality in experimental dengue virus infectian. FEMS Immunol Med Microbiol 2003;35(1):33-42.

32.    Huang YH, Lei HY, Liu HS, Lin YS, Liu OO, Yeh TM. Dengue virus infects human endathe-lial cells and induces IL-6 and IL-8 praductian. Am J Trop Med Hyg 2000;63(1-2):71-75.

33.    Bazza FA, Oruz OG, Zagne SM, Azereda EL, Nagueira RM, Assis EF, et al. Multiplex cytakine prafile fram dengue patients: MIP-1beta and IFN-gamma as predictive factars far severity. BMC Infect Dis 2008;8:86.

34.    Srikiatkhacharn A, Green S. Markers af Dengue Disease Severity. Current Topics in Micro-biology and Immunology 2009;1(2):68-79.

35.    Zivna I, Green S, Vaughn DW, Kalayanaraaj S, Stephens HA, Ohandanayingyang D, et al. T cell respanses ta an HLA-B*07-restricted epitape an the dengue NS3 pratein carrelate with disease severity. J Immunol 2002;168(11):5959-5965.

36.    Restrepa BN, Ramírez R, Arboleda M, Álvarez G, Ospina M, Díaz FJ. Levels af cytakines in twa ethnic graups with dengue virus infectian. Am J Trop Med Hyg 2008;79(5):673-677.

POSGRADOS CLÍNICOS

Universidad

Surcolombiana

ESPECIALIZACIÓN EN PEDIATRÍA

Registro ICFES No. 111456180924100111400 Creada mediante acuerdo Consejo Superior Universitario No. 034 del 29-05-1996

Denominación Académica:    Programa de Especialización en Pediatría

Modalidad:    Presencial

Duración:    3 años (6 semestres)

Cupos:    3 Anual

Título:    Especialista en Pediatría

ESPECIALIZACIÓN EN CIRUGIA GENERAL

Registro ICFES No. 111456170004100111400 Creada mediante acuerdo Consejo Superior Universitario No. 035 del 29-05-1996

Denominación Académica: Modalidad:

Duración:

Cupos:

Título:

Programa de Especialización en Cirugía General Teórico - Presencial 4 años (8 semestres)

2 Anuales

Especialista en Cirugía General

ESPECIALIZACIÓN EN GERENCIA DE SERVICIOS DE SALUD Y SEGURIDAD SOCIAL

Creada mediante acuerdo Consejo Superior Universitario No. 021 del 10-07-1995 SNIES No. 3503, Registro calificado de calidad Res. No 450 de febrero 5 de 2008

Ministerio de Educación Nacional.

Denominación Académica: Programa de Especialización en Gerencia de Servicios de salud

y Seguridad Social Modalidad:    Semipresencial

Duración:    3 semestres

Cupos:    25 Anuales

Título:    Especialista en Gerencia de Servicios de salud y Seguridad Social

1

Grupa de Parasitalagía y Medicina Trapical, Pragrama de Medicina, Facultad de Salud, Universidad Surcalambiana, Neiva, oalambia.

Oarrespanding authar: jradriguez@usca.edu.ca