Upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in renal tissue in severe dengue in humans: Effects on endothelial activation/dysfunction

Abstract INTRODUCTION: Dengue is an important mosquito-borne disease in tropical and subtropical regions. Adhesion molecules have not been systematically characterized in the renal tissue of patients with severe dengue (SD). The objective of this study was to detect viral antigens in samples from patients that evolved with SD, correlating with the expression of ICAM-1, VCAM-1, VE-cadherin, and E-selectin to contribute to a better understanding of the pathophysiology of SD. METHODS: Kidney specimens from patients with SD were selected according to clinical and laboratorial data and submitted to histological and immunohistochemistry analysis. A semiquantitative evaluation was performed considering positive immunostaining in 20 glomeruli. RESULTS: Viral antigens were mainly detected in distal tubules. The intense immunostaining of VCAM-1 and ICAM-1 was observed. The expression of E-selectin was discrete, and VE-cadherin expression varied from mild to moderate. VCAM-1 was slightly intense in the glomerular capsule; the expression of ICAM-1 was diffuse. E-selectin was diffuse, and VE-cadherin varied from mild to moderate. The most frequent histological findings were glomerular congestion, mild glomerulitis, acute renal injury, and glomerular atrophy. CONCLUSIONS: The results appear to demonstrate an imbalance between vascular endothelial permeability regulating events in renal lesions in SD. The increase in the expression of ICAM-1 and VCAM-1 is an in-situ indicator of higher permeability with a consequent influx of cells favoring the inflammation of the endothelium. These molecules are important in the pathophysiology of the disease and provide the possibility of developing new markers for the evaluation, clinical follow-up, and therapeutic response of patients with SD.

Relationships of interleukin-10 with the regulatory T cell ratio and prognosis of cervical cancer patients

OBJECTIVE: This study investigated serum interleukin-10 (IL-10) levels, changes in peripheral blood CD4+CD25+ regulatory T cell (PBCDT) ratios, and the prognosis of cervical cancer (CC) patients. METHODS: Seventy patients with CC composed the observation group, and 70 healthy subjects composed the control group. The PBCDT ratios in the CC patients and healthy subjects were calculated. Serum IL-10 levels were detected with a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The PBCDT ratio was higher in the patients with active CC [12.16±2.41%] than in the control subjects [6.34±1.05%]. Serum IL-10 levels were higher in the patients with CC [384±106 pg/ml] than in the control subjects [104±50 pg/ml]; the differences in both PBCDT ratio and IL-10 level were statistically significant (p<0.01). Serum IL-10 levels were positively correlated with PBCDT ratios (r=0.375, p<0.05). The 5-year patient survival rate was significantly higher in the low serum IL-10 group (64.2%) than in the high serum IL-10 group (42.8%, p=0.012). CONCLUSIONS: PBCDT ratios and serum IL-10 levels are related to CC activity. These factors are reciprocally related and influence one another, and both are involved in the development and progression of CC. Low IL-10 expression is beneficial regarding the survival of patients with CC.

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria / Avanços técnicos no diagnóstico e no monitoramento de hemoglobinúria paroxística noturna por citometria de fluxo

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones.

RESUMO Objetivo: Discutir as melhorias técnicas no diagnóstico e no acompanhamento laboratorial de hemoglobinúria paroxística noturna para a validação da técnica de citometria de fluxo de alta sensibilidade. Métodos: Estudo retrospectivo, que envolveu a análise de dados laboratoriais de 745 pacientes com hipótese diagnóstica e/ou acompanhamento de hemoglobinúria paroxística noturna por citometria de fluxo. Resultados: Os avanços técnicos não só reduziram o custo do ensaio, mas também melhoraram a identificação e a resolução da citometria de fluxo para a detecção de clone hemoglobinúria paroxística noturna. Conclusão: A citometria de fluxo de alta sensibilidade possibilitou a identificação do tipo e do tamanho de clone de hemoglobinúria paroxística noturna, especialmente em amostras com pequeno clone.

Ginandroblastoma multiloculado, negativo para CD99 en una niña de 12 años: caso clínico / A twelve-years-old girl with multiloculated gynandroblastoma CD99 negative. Case report

Los ginandroblastomas son tumores del ovario extremadamente raros, los cuales comparten componentes de células de la granulosa y de células de Sertoli/Leydig. Se describe un caso de una niña de 12 años, quien presenta hemorragia uterina anormal y sensación de masa intraabdominal de crecimiento progresivo asociado a menorragia, niveles de CA-125 en 60,4 UI/mL y estudios de extensión que reportan masa quística en ovario izquierdo, manejada con ooforectomía. El estudio anatomopatológico muestra un tumor multiloculado lleno de material seroso, abundantes cuerpos de Call-Exner y 45% de células de Sertoli/Leydig. La inmunohistoquímica reveló inmunorreactividad para inhibina, calretinina y pCK, mientras que los marcadores CD99 y AE1/AE3 fueron negativos. Se trata del primer reporte de caso sobre un ginandroblastoma multiloculado, negativo para CD99 en una niña de 12 años, estudio que plantea un abordaje sistemático para los tumores de las células de los cordones sexuales.

The ginandroblastoma is an extremely rare ovarian tumor which shows components of granulosa cells and Sertoli/Leydig cells. We describe a case of a twelve-years-old girl who presented abnormal uterine bleeding and progressively growing intra- abdominal mass associated with menorrhagia, CA-125 60.4 UI/mL and extension studies reporting cystic mass in the left ovary. She underwent oophorectomy. Pathological study shows a multilocular tumor filled with serous material. Many Call-Exner bodies were observed in the histopathological analysis, 45% of Sertoli/Leydig cells. Immunohistochemistry was reactive for inhibin, calretinin and pCK while AE1/AE3 and CD99 markers were negative. This is the first case report about a multiloculated gynandroblastoma, negative for CD99 in a 12-years-old girl. Thus, the study of this clinical case represents a systematic approach for tumors of the sex cord cells.

Evaluation of Toll-like, chemokine, and integrin receptors on monocytes and neutrophils from peripheral blood of septic patients and their correlation with clinical outcomes

Recognition of pathogens is performed by specific receptors in cells of the innate immune system, which may undergo modulation during the continuum of clinical manifestations of sepsis. Monocytes and neutrophils play a key role in host defense by sensing and destroying microorganisms. This study aimed to evaluate the expression of CD14 receptors on monocytes; CD66b and CXCR2 receptors on neutrophils; and TLR2, TLR4, TLR5, TLR9, and CD11b receptors on both cell types of septic patients. Seventy-seven septic patients (SP) and 40 healthy volunteers (HV) were included in the study, and blood samples were collected on day zero (D0) and after 7 days of therapy (D7). Evaluation of the cellular receptors was carried out by flow cytometry. Expression of CD14 on monocytes and of CD11b and CXCR2 on neutrophils from SP was lower than that from HV. Conversely, expression of TLR5 on monocytes and neutrophils was higher in SP compared with HV. Expression of TLR2 on the surface of neutrophils and that of TLR5 on monocytes and neutrophils of SP was lower at D7 than at D0. In addition, SP who survived showed reduced expression of TLR2 and TLR4 on the surface of neutrophils at D7 compared to D0. Expression of CXCR2 for surviving patients was higher at follow-up compared to baseline. We conclude that expression of recognition and cell signaling receptors is differentially regulated between SP and HV depending on the receptor being evaluated.

Angiogenic potential of human umbilical cord blood ex vivo expanded CD 133* stem cells in chronic hepatic fibrosis of murine schistosomiasis

As a source of hematopoietic stem cells [HSCs], umbilical cord blood [UCB] has the advantages of speed of availability, tolerance of more than one HLA mismatch, and a low incidence of severe graft-versus-host disease [GVHD]. Hence, it represents a promising, alternative non-costly and non-invasive source for prospective stem cell based therapy. In this study we investigated the angiogenic potential of ex vivo expanded human umbilical cord blood CD 133* stem cells transplanted into mice with chronic hepatic fibrosis induced by Schistosomiasis infection. Histopathological, ultrastructural and immunohistochemical analysis of mice liver sections were done to detect specific human angiogenic markers. Umbilical cord blood was obtained from healthy pregnant females after delivery and mononuclear cells were collected by density gradient using Ficoll Hypaque. Enrichment for the CD 133* stem cells was done by positive selection using the Magnetic Activated Cell Sorting system and magnetic microbeads. Cells were cultured in prirnaly ex vivo expansion medium for three weeks. Flowcytomeric analysis of the cultured cells was done in each step to identify the CD 133* cells. Schistosomiasis was induced in Swiss Albino mice by intradermal injection of schistosoma cercariae. Twenty two weeks post schistosoma infection a total of 0.3 x 106 human CD 133* stem cells were injected intrahepatically in mice. Accordingly, mice were divided into three groups: Group 1 [infected, transplanted]; Group 2 [infected controls] and Group 3 [healthy, transplanted]. All mice were sacrificed 3 wks after cell transplantation was done in groups I and 3. Histopathology and Electron microscopy showed an obvious increase in the capillary network and the small blood vessels and a decrease in the fibrosis known for this stage of the disease. By immunohistochemical analysis the cellular constituents of these newly formed blood vessels showed positive expression of the human-specific angiogenic markers CD31, CD34 and von Willebrand Factor [vWF]. Few hepatocyte like polygonal cells showed positive expression of human Vascular Endothelial Growth Factor [VEGF] and inducible Nitric Oxide Synthase [iNOS]. Ex vivo expanded CD 133* human stem cells incorporate into the liver of schistosoma infected mice enhancing local angiogenesis and hepatic neovascularization. These preliminary results obtained suggest a dual benefit of CD 133* cells in cell therapy in hepatic diseases based on its capability of hematopoietic and endothelial differentiation. We suggest that the CD 133* cells contribute to repair in a paracrine manner by creating a permissive environment that enables rapid proliferation and survival of damaged cells rather than through direct differentiation to hepatocytes

Recent advances in management of acute myeloid leukemia (AML).

Acute myeloid leukemia (AML) is the most common childhood malignancy. AML has therapeutically been difficult to treat. In 2001, the World Health Organization (WHO), in conjunction with the Society for Hematopathology and the European Association of Hematopathology, published a new classification for myeloid neoplasms. A number of chromosomal abnormalities are used to predict outcome and stratify therapeutic risk groups in children with AML. Recently, alterations in receptor tyrosine kinases, tyrosine phosphatases and in oncogenes such as RAS have been implicated in the pathogenesis of AML. This article aims to review the recent development in diagnosis, treatment and monitoring of AML. Better understanding of the molecular pathogenesis of AML has led to the development of target-specific therapies. Some of the new classes of drugs include monoclonal antibody directed against the CD33 antigen, farnesyltransferase inhibitors (FTI), and FMSlike tyrosine kinase 3 (FLT3) inhibitors. The role of allogenic SCT, particularly whether it should be done during first CR or reserved for second remission, remains the most controversial issue in pediatric AML. There is a need of collaboration with international pediatric cooperative oncology groups and definitive clinical trials in order to establish use of these newer molecules in pediatric populations.

Relation between maternal plasma concentration of serum endoglin in patients with IUGR and preeclampsia: correlation with doppler study

The aim of the study was to compare between the rise in serum endoglin [sEng] in pregnant women with normotensive IUGR and preeclamptic pregnant women with and without IUGR and to compare between serum placental growth factor [PIGF] and endoglin levels in serum of pregnant patients with IUGR. Prospective analytic comparative study. The study includes group [1] 17 cases with manifest preeclampsia without IUGR, group [2] 11 cases with preeclampsia and IUGR, group [3] 12 normotensive pregnancies with IUGR and 15 gestational age matched control. sEng and PIGF were determined by ELISA, Doppler examination of the umbilitcal vessels in the form of PI and S/D ratio were done. The three groups show significantly higher sEng concentration compared to the control group [32.2 +/- 6.0 ng/ml, 42.3 +/- 10.5 ng/ml, 24.5 +/- 7.1 ng/ml and 12.2 +/- 3.6 ng/ml in the 4 groups respectively, p<0.001]. The three groups showed significantly lower level of PIGF [45.4 +/- 20.4 pg/ml, 23.3 +/- 12.1 pg/ml, 73.1 +/- 54.7 pg/ml and 111.1 +/- 58.4 pg/ml in the 4 groups respectively. P<0.003]. There was no significant difference between sEng in the three groups. Significant difference was found for serum levels of PIGF between normotensive IUGR and the other 2 groups. There was a significant positive correlation between sEng levels and S/D ratio [r= .939 p< 0.001] and PI [r=.695 p<0.001] of the umbilical vessels in group of preeclampsia without IUGR and insignificant correlation between serum levels of PIGF and Doppler study of the umbilical vessels in the same group. sEng increased in all cases with placental pathology even in normotensive IUGR, while PIGF has the reverse relationship, it decreased in placental pathology with negative relationship with severity. The correlation between maternal serum levels of sEng and PIGF and Doppler ultrasound indices of umbilical arteries in pre-eclampsia and IUGR reflect the severity of the disorders

Macrophage Activation Syndrome in a Child with Systemic Juvenile Rheumatoid Arthritis

Macrophage activation syndrome (MAS) is a rare and potentially fatal complication of rheumatic disorders in children. We describe a 13-month-old boy in whom MAS developed as a complication of systemic juvenile rheumatoid arthritis (S-JRA). He suffered from fever and generalized rash followed by multiple joints swelling for four months before admission. Physical examination revealed cervical lymphadenopathy and hepatosplenomegaly. Laboratory findings were: abnormal liver enzymes, increased triglyceride and ferritin levels, coagulopathies resembling disseminated intravascular coagulation, anemia and thrombocytopenia. Hyperplasia of hemophagocytic macrophages was remarkable in his bone marrow. Methylprednisolone and cyclosporin therapy resulted in clinical and laboratory improvements. This is the third case of MAS associated with S-JRA in Koreans, and the first one, in which hemophagocytic macrophages were proven in bone marrow.

Determination of laboratory reference ranges for lymphocyte immunophenotype subsets in healthy adult Japanese using flow cytometry.

This study represents a comprehensive evaluation of normative values for lymphocyte immunophenotype subsets using flow cytometry techniques in a Japanese population. Lymphocyte reference ranges were determined for percentage and absolute count of T, B, and NK cells in healthy adult Japanese using an extensive two-color immunophenotyping panel and consistently applied quality control methodology. Reference values were also determined for activation markers on CD3+ lymphocytes CD3+/CD25+, CD3+/CD38+ and CD3+/HLA-DR+. Differences in age and gender were observed for specific lymphocyte subsets. Comparison of the Japanese study with a Thai multi-center study that used similar methodology also demonstrated ethnic differences in lymphocyte reference ranges. The results in this study strongly suggest that reference values derived from studies in one population may not be applied to another population even when similar protocols for reagents, instruments and procedures are used although such studies do appear useful for epidemiological comparisons.

Pilot neonatal screening program for lysosomal storage disorders, using lamp-1.

We have demonstrated that the lysosome associated membrane protein (LAMP-1) is elevated in plasma from approximately 70% of lysosomal storage disorder patients. As part of the development of a newborn screening program for lysosomal storage disorders we have developed a first tier screening assay based upon the level of LAMP-I in blood spots taken from newborn Guthrie cards. To determine the effectiveness of the first-tier marker a prospective pilot Guthrie neonatal screening program for the identification of LSD was commenced in April 1998. Prior to commencement of the pilot program ethical approval was obtained and information leaflets regarding the neonatal screening of LSD were distributed to parents at the time of their infant's Guthrie collection. The LAMP-1 assay utilizes a chicken polyclonal and a mouse monoclonal in a sandwich time resolved fluorescent immunoassay. LAMP-1 blood-spot calibrators and quality control specimens were developed and shown to be stable and reproducible. To date 11,183 infants have been screened using LAMP-1. The population distribution is described with a median and 98th percentile of 220pg/l whole blood and 483microg/l whole blood respectively. Acceptable CV% for intra and inter assay of 8.9% and 10% respectively were obtained.

Newborn screening for lysosomal storage disorders.

Lysosomal storage disorders (LSD) represent a group of over 40 distinct genetic diseases with a total incidence of approximately 1:7,000 births. Bone marrow transplantation and enzyme replacement therapy are currently in use for the treatment of some disorders and new forms of enzyme and gene replacement therapy are actively being researched. The effectiveness of these therapies, particularly for the LSD involving the central nervous system and bone pathology, will rely heavily upon the early diagnosis and treatment of the disorder, before the onset of irreversible pathology. In the absence of a family history the only practical way to detect these disorders will be by a newborn screening program. One common feature of these disorders is an increase in the number and size of lysosomes within the cell from approximately 1% to as much as 50% of total cellular volume. Associated with this, is a corresponding increase in some lysosomal proteins. We propose that the measurement of one or more of these proteins in blood spots taken from Guthrie cards, will form the basis of a newborn screening program, for the detection of all LSD. We have identified a number of lysosomal proteins as potential markers for LSD. The level of these proteins has been determined in blood spots taken from Guthrie cards and in plasma samples from over 300 LSD affected individuals representing 25 disorders. Based on these results we have proposed a strategy for a newborn screening program involving a two tier system, utilizing time resolved fluorescence immunoquantification of the protein markers in the first tier, followed by tandem mass spectrometry for the determination of stored substrates in the second tier assays.

Umbilical cord blood as a source of CD34 positive haematopoietic stem cells.

In this paper we have used a monoclonal antibody to CD34 an antigen expressed solely on stem cells, and stem cell colony assays to show that umbilical cord blood has nearly the same number of functional stem cells as compared to normal bone-marrow. The number of CD34+ve cells in cord blood being 2 to 2.7 per cent, whereas bone-marrow had 3 to 3.5 per cent. The multi-potent colony forming cells (CFU-GEMM) were 60 +/- 18 in cord blood per 2 x 10(5) mononuclear cells (MNCs), whereas normal bone-marrow had 70 +/- 10 per 2 x 10(5) MNCs. Enrichment of these stem cells on Percoll gradients was successful for normal bone-marrow but not for cord blood.

Acute non-lymphocytic leukemia with expression of surface antigen CD7--morphological, cytochemical, immunological and ultrastructural features in eight patients.

Of late, there has been an increase in the number of acute leukemias coexpressing markers believed to be restricted to a single lineage. Eight patients with ANLL whose blast coexpressed the T cell associated CD7 antibody were identified among 462 consecutive ANLL cases. Seven had FAB defined AML according to morphocytochemical criteria, whereas one patient was classified as MO on the basis of ultrastructural studies. The incidence of CD7 positivity was particularly significant in the less differentiated sub-types MO and M1 compared to other FAB sub-groups. Detailed long term studies will be required to realize their biological and clinical significance.

Age-associated changes of memory (CD45RO+) and naive (CD45R+) T cells

The total number of lymphocytes and the percentage of CD45RO+ (putative memory T cell) and CD45R+ (putative naive T cell) were determined in 15 cord blod samples, 66 healthy children ranging in a age from 1 to 18 years, 16 adults (23-59 years) and 16 aged individuals (60-96 years). The total number of lymphocytes decreased with age and reached the adult range in children to the adult group,white the percentage of CD45RO+ Tcells was low in cord blood and increased with age.No significant difference was observed between the adult and the aged groups for either lymphocyte subset. These data support the view that CD45RO+ and CD45R+ T-cell subsets represent maturational stages of T cells