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Abstract Background Extranodal NK/T-cell lymphoma, nasal type (ENKTL) is not common worldwide, but it is the most common T- and NK-cell lymphomas in many Asian countries. Immunophenotypic profiles were studied based on limited series. The authors, therefore, studied on ENKTL according to characterize immunophenotypic profiles as well as the distribution of EBV subtype and LMP-1 gene deletion. Methods By using tissue microarray (TMA), immunohistochemical study and EBV encoded RNA (EBER) in situ hybridization were performed. T-cell receptor (TCR) gene rearrangement, EBV subtyping, and LMP-1 gene deletion were studied on the available cases. Results There were 22 cases eligible for TMA. ENKTL were positive for CD3 (91%), CD5 (9%), CD7 (32%), CD4 (14%), CD56 (82%), TIA-1 (100%), granzyme B (95%), perforin (86%), CD45 (83%), CD30 (75%), Oct2 (25%), and IRF4/MUM1 (33%). None of them was positive for βF1, CD8, or CD57. TCR gene rearrangement was negative in all 18 tested cases. EBV was subtype A in all 15 tested cases, with 87% deleted LMP-1 gene. Cases lacking perforin expression demonstrated a significantly poorer survival outcome (p = 0.008). Conclusions The present study demonstrated TIA-1 and EBER as the two most sensitive markers. There were a few CD3 and/or CD56 negative cases noted. Interestingly, losses of CD45 and/or CD7 were not uncommon while Oct2 and IRF4/MUM1 could be positive in a subset of cases. Based on the present study in conjunction with the literature review, determination of PCR-based TCR gene rearrangement analysis might not be a useful technique for making diagnosis of ENKTL.

Abstract Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan) has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR) followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM). The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma. Introduction Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population 1. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year 2. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal lymphoproliferation 34. Analyzing DNA extracted from formalin-fixed, paraffin-embedded tissues by multiplex PCR techniques is more rapid, accurate and highly sensitive. Measuring the size of the amplicon from PCR analysis could be used to diagnose malignant lymphoma with monoclonal pattern showing specific and distinct bands detected on acrylamide gel electrophoresis. However, this technique has some limitations and some patients might require a further confirmation test such as GeneScan or fragment analysis 56. GeneScan technique or fragment analysis reflects size and peak of DNA by using capillary gel electrophoresis. This technique is highly sensitive and can detect 0.5-1% of clonal lymphoid cells. It measures the amplicons by using various fluorescently labeled primers at forward or reverse sides and a specific size standard. Using a Genetic Analyzer machine and GeneMapper software (Applied Bioscience, USA), the monoclonal pattern revealed one single, sharp and high peak at the specific size corresponding to acrylamide gel pattern, whereas the polyclonal pattern showed multiple and small peak condensed at the same size standard. This technique is the most sensitive and accurate technique; however, it usually requires high technical experience and is also of high cost 7. Therefore, rapid and more cost effective technique are being sought. LightCycler PCR performs the diagnostic detection of amplicon via melting curve analysis within 2 hours with the use of a specific dye 89. This dye consists of two types: one known as SYBR-Green I which is non specific and the other named as High Resolution Melting analysis (HRM) which is highly sensitive, more accurate and stable. Several reports demonstrated that this new instrument combined with DNA intercalating dyes can be used to discriminate sequence changes in PCR amplicon without manual handling of PCR product 1011. Therefore, current investigations using melting curve analysis are being developed 1213. In this study, three different techniques were compared to evaluate the suitability of LightCycler PCR with HRM as the clonal diagnostic tool for IgH gene rearrangement in B-cell non-Hogdkin lymphoma, i.e. thermocycler PCR followed by heteroduplex analysis and PAGE, GeneScan analysis and LightCycler PCR with HRM.

Plumericin, an iridoid lactone, was isolated with relatively high yield from Momordica charantia vine using the supercritical fluid extraction (SFE) and the separation box (Sepbox) comprising dual combination of high-performance liquid chromatography and solid phase extraction. This compound showed antibacterial activity against Enterococcus faecalis and Bacillus subtilis with minimum inhibitory concentration (MIC) values better than cloxacillin. Plumericin potently inhibited proliferation of two leukemic cancer cell lines: they were acute and chronic leukemic cancer cell lines, NB4 and K562, with the effective doses (ED50) of 4.35 ± 0.21 and 5.58 ± 0.35 μg/mL, respectively. In addition, the mechanism of growth inhibition in both cell lines was induced by apoptosis, together with G2/M arrest in K562 cells. [ABSTRACT FROM AUTHOR]

AML1/RUNX1, a member of the core binding factor (CBF) family stimulates myelopoiesis and lymphopoiesis by activating lineage-specific genes. In addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells via transactivation. We now found that AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation. Western blotting and a gel shift/supershift assay demonstrated that endogenous CBF DNA binding and AML1 levels were increased 2-4-fold in S and G2/M phase cells compared with G1 cells. In addition, G1 arrest induced by mimosine reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or AML1-ER demonstrated a significant reduction in G1 phase cells, whereas levels of exogenous DNA binding domain alone were constant, lending support to the conclusion that regulation of AML1 protein stability contributes to cell cycle variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation was independent of cell cycle phase, and an AML1 mutant lacking two ERK phosphorylation sites was still cell cycle-regulated. Inhibition of AML1 activity with the CBFβ-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA expression, and AML1 bound and activated the cyclin D3 promoter. Signals stimulating G1 to S cell cycle progression or entry into the cell cycle in immature hematopoietic cells might do so in part by inducing AML1 expression, and mutations alterlng pathways regulating variation in AML1 stability potentially contribute to leukemic transformation. [ABSTRACT FROM AUTHOR]

Breast cancer is the most frequent type of cancer diagnosed among women worldwide and also in Thailand. Estrogen and estrogen receptors exert important roles in its genesis and progression. Several cytokines have been reported to be involved in the microenvironment that promotes distant metastasis via modulation of immune and inflammatory responses to tumor cells. Estrogen receptor genetic polymorphisms and several cytokines have been reported to be associated with breast cancer susceptibility and aggressiveness. To investigate roles of genetic polymorphisms in estrogen receptor alpha (ESR1) and interleukin 6 (IL6), breast cancer patients and control subjects were recruited from the Division of Head, Neck and Breast Surgery (Siriraj Hospital, Bangkok, Thailand). Polymorphisms in ESR1 (rs3798577) and IL6 (rs1800795 and rs1800797) were evaluated by real-time PCR in 391 breast cancer patients and 79 healthy controls. Associations between genetic polymorphisms and clinicopathological data were determined. There was no association between genetic polymorphisms and breast cancer susceptibility. However the ESR1 rs3798577 CT genotype was associated with presence of lymphovascular invasion (OR=2.07, 95%CI 1.20-3.56, p=0.009) when compared to the TT genotype. IL6 rs1800795 CC genotype was associated with presence of extranodal extension (OR= 2.30, 95%CI 1.23-4.31, p=0.009) when compared to the GG genotype. Survival analysis showed that IL6 rs1800797 AG or AA genotypes were associated with lower disease-free survival. These findings indicate that polymorphisms in ESR1 and IL6 contribute to aggressiveness of breast cancer and may be used to identify high risk patients.

Polymorphisms in the promoter and 5' untranslated region of vascular endothelial growth factor (VEGF) have been associated with VEGF levels. To investigate the role of VEGF polymorphisms in breast cancer, the VEGF ‑2578C/A, ‑1498C/T, ‑1154G/A and ‑634G/C polymorphisms were genotyped in 483 breast cancer patients and 524 healthy controls. VEGF mRNA levels in breast cancer tissue were determined using semi‑quantitative RT‑PCR. The genotypes, ‑634G/C and ‑634C/C, were associated with an increased risk for breast cancer when compared with the ‑634G/G genotype. The VEGF ‑634G/C genotype was associated with tumor size >20 mm, perineural invasion and stage II‑IV. Individuals with ‑634C/C had lower disease‑free survival. Patients with the VEGF ‑634C/C genotype exhibited the highest VEGF mRNA levels. High VEGF mRNA expression correlated with tumor size >20 mm, presence of lymphovascular invasion and axillary nodal metastasis. These observations suggested that VEGF ‑634G/C polymorphisms have a significant role in breast cancer susceptibility and aggressiveness.

Previous report showed the high potent antiproliferative effect of the methanolic part extracted from the aerial parts of Pouzolzia indica on NB4 and HT93A acute leukemic cell lines with the IC50 values of 28.5 and 49.8  μ g/mL, respectively. The bioassay-guided fractionation of the methanolic part gave 5 fractions, that is, FFI-FFV. FFII, FFIII, and FFIV inhibited the above leukemic cell lines with the IC50 values of 15.1 (FFII), 14.4 (FFIII), 32.1 (FFIV), and 31.0 (FFII), 9.7 (FFIII), 10.5 (FFIV)  μ g/mL, respectively. The compounds in these fractions were isolated using chromatographic technique. FFII contained friedelin 1, 28-hydroxy-3-friedelanone 2, and 7-methoxy-coumarin 3. FFIII contained 6, 7-dimethoxy-coumarin 4, scopoletin 5, methyl caffeate 6. FFIV contained sitosteryl glucoside 7 and a supposed glycosphingolipid 8. The chemical structures were elucidated by spectroscopic methods.

The present study was conducted to investigate the morphological and structural changes of Acanthamoeba cysts after being treated with various concentrations of Pouzolzia indica methanolic extract fraction 3 (methanol eluted) and Virkon solution. Changes in the Acanthamoeba cysts were detected by light microscopy, scanning electron microscopy and transmission electron microscopy. The results show Acanthamoeba cysts were killed by Pouzolzia indica methanolic extract fraction 3 at a concentration of 1:8 and by Virkon solution at a concentration of 0.25%, with a minimal cysticidal concentration (MCC) by 24 hours. Both agents caused similar structural damage to Acanthamoeba cysts in the same sequence. Step by step structural alterations occurred within the cyst. First, the cyst shrank, collapsed and had clumping of cytoplasmic stuctures inside the cyst walls. Second, the cysts began to bulge, swell, have a decrease in wrinkles in the cyst walls and spill the cytoplasmic contents into the environment. Finally, the cyst walls broke into small pieces. This study may be beneficial to compare with future studies of pharmaceutical agents against Acanthamoeba keratitis.

Objective: To compare the minimal cysticidal concentration (MCC) between Pouzolzia indica methanolic extract fraction 2 and Povidone-lodine (PVP-I) on the Acanthamoeba cyst and to illustrate the morphological changes of the cyst after being treated by light and electron microscopies., Material and Method: Acanthamoeba spp were isolated from patients with Acanthamoeba keratitis and cultured on a non-nutrient agar plate (NNA) seeded with heat killed Escherichia coli (NNA-E.coli) at 37 degrees C for 7 days, adjusted to a final concentration of 10(4) cysts/ml. Several concentrations of PVP-I and fraction 2 of Pouzolzia indica methanolic extract were tested to find the minimal cysticidal concentrations (MCC) of both agents, at these concentrations there was no viable cyst which was confirmed by no excystment after further incubation for 7 days. The cysts were prepared for routine transmission and scanning electron microscopic studies., Results: Structural damages of the treated cysts by MCC of PVP-I and fraction 2 of Pouzolzia indica methanolic extract showed a series of damages. Starting from shrinkage, destruction or rupture of the cyst walls and opercula, withdrawal of the cytoplasm or edema cyst by the outside solution passed through the damaged wall caused a decrease in wrinkle ridges of the ectocyst. Then the cyst was ripped and torn into small pieces, Conclusion: MCC of PVP-I solution and the fraction 2 of Pouzolzia indica methanolic extract were 0.04% and 1:4, respectively. The structural damages were somewhat similar, such as the shrinkage, ruptured cyst wall and opercula, edema and end by breaking up of the cyst wall and degeneration of the inside cytosol. Pouzolzia indica may be modified as an effective disinfectant solution for a contact lens case if the active ingredients are more purified.

The root extract of Trichosanthes cucumerina L. and bryonolic acid (1), its main constituent, as well as the fruit juice and cucurbitacin B (3), its main constituent, were tested for cytotoxicity against four human breast cancer cell lines (SKBR3, MCF7, T47D, and MDA-MB435), two lung cancer cell lines (A549 and SK-LU1), and one colon cancer cell line (Caco-2). The root extract had higher IC (50) values than bryonolic acid (1) against three breast cancer cell lines (MCF7 = 267/121, T47D = 316/124, MDA-MB435 = 140/90 microL/mL) and one lung cancer cell line (A549 = 106/100 microL/mL). The fruit juice also had higher IC (50) values than cucurbitacin B (3) against the four breast cancer cell lines (131/73, 375/35, 249/60, and 156/26 microL/mL, respectively) and one lung cancer cell line (141/41 microL/mL) as shown above, as well as against the colon cancer cell line (101/1.5 microL/mL). However, the root extract inhibited SK-LU1 more strongly than did the fruit juice, cucurbitacin B (3), and bryonolic acid (1) (149/169/180/>500 microL/mL, respectively). The root extract inhibited the two lung and three breast cancer cell lines (SKBR3, MDA-MB435, and MCF7) more strongly than the fruit juice. Bryonolic acid (1) inhibited MDA-MB435 somewhat better than the other tested human cancer cell lines. The fruit juice inhibited the colon cancer cell line (Caco-2) more strongly than the root extract. Cucurbitacin (3) inhibited human cancer cell lines, especially Caco-2, much more strongly than bryonolic acid (1). In addition to bryonolic acid (1), bryononic acid (2), cucurbitacin B (3), and dihydrocucurbitacin B (4) also were isolated from the root extract.

Background: Anti-Ro antibody may directly react against either Ro60 or Ro52 or both antigens. To be more applicable for routine laboratory practice, the specific antigen type for antibody detection should be identified before test application., Objective: Investigate the prevalence of 60 kDa and 52 kDa Ro/SS-A antibodies in Thai patients' sera in Siriraj Hospital., Material and Method: Specimens for anti-Ro were requested between June and December 2005. They were tested with EUROLINE test kit for prevalence determination. The principle of the test is a qualitative in-vitro-assay that contains test strips coated with parallel lines of 14 highly purified antigens. Of 84 specimens requested for anti-Ro antibody, 76 were collected and tested with the EUROLINE test kits and eight were excluded due to inadequacy., Results: The prevalence of anti-Ro60 and anti-Ro52 of all sera tested for anti-Ro by EUROLINE test kit were 30% (95% CI: 20-40%) and 26% (95% CI: 16-36%), respectively; and, those in anti-Ro positive Thai sera were 82% (95% CI: 68-96%) and 71% (95% CI: 54-88%), respectively. The prevalence of anti-Ro52 alone in anti-Ro positive Thai sera and all specimens requested for anti-Ro was about 18% (95% CI: 4-32%) and 7% (95% CI: 1-13%), respectively. The agreement and Kappa value between the two methods were 0.9 and 0.77, respectively. The study suggests that the test for anti-Ro detection should provide both Ro 60 and Ro 52 antigens., Conclusion: The prevalence of both anti-Ro 60 and anti-Ro 52 were quite common, therefore, the test for this specific antibody should provide both antigens for antibody detection.

Medicinal plants have long been used and prescribed in Thailand for centuries. Some of them have been used for treating various diseases including infectious diseases. Pouzolzia pentandra Benn., Gelonium multiflorum A. Juss., Erycibe elliptilimba Merr. and Chun., Balanophora abbreviate Bl. are Thai medicinal plants from the Thai pharmacopoeia that have been prescribed for treating unknown fevers including some specific infectious diseases. This investigation demonstrated the effects of these Thai medicinal plants for their antibacterial activities by using the macrodilution assay. Based on the present study, the water methanol fraction (fraction 2) of Balanophora abbreviate Bl. showed the antibacterial activity at the MIC level of 250 microg/ml but the activity was bacteriostatic in its effects. Therefore, the use of these medicinal plants in controlling fever and infectious diseases appears to be justified and further investigations may be required to obtain more information.

Herbs have been considered natural and valuable sources for anticancer drug discovery. Herbal medicine has been prescribed in many countries over centuries for treating various diseases including infectious and malignant diseases. Nowadays, many of the drugs that have been used for treatment of malignant diseases are derived from natural products such as Taxol, a natural product isolated initially from Pacific Yew (Taxus brevifolia). This review article describes research on molecular mechanisms, especially cytotoxic effect of natural products from plant sources, primarily preclinical studies, involving human lung cancer cells in vitro for providing more knowledge and issues for potential drug development from medicinal herbs in the future.