Panleucogating as an accurate and affordable flow cytometric protocol to analyse lymphocyte subsets among HIV-positive patients on HAART treatment in Mozambique

In Africa tens of millions of people are HIV+. Prevention alone is not effective, and needs to be coupled with anti-retroviral treatment (HAART). Laboratory tests as CD4+ T cell count are fundamental tools in HIV disease monitoring, but they require costly equipment, reagents and specialised manpower. The goal of this study was to minimise and optimise the reagents needed for a reliable routine CD4+ cell count in a resource-poor setting (Mozambique). Panleucogating protocol (PLG), requires two antibodies only, CD45 and CD4, or three if CD8 is requested for special clinical reasons. PLG was compared with the current protocol used in two Mozambique hospitals, based on FSC/SSC gating and CD3/CD4/CD8 staining. 189 samples from HIV+ patients, included in the Community of Sant'Egidio's DREAM program and on HAART were processed with both protocols. The overall correlation of the lymphocyte subsets measurements was satisfactory, with r2 always >0.96. The Bland-Altman analysis of CD4+ cell count showed a negative bias when CD4+ cells were <15%, due to the imprecise FSC/SSC gating used previously. When CD4+ cells were >15% the negative bias tended to zero, further confirming the better quality of the PLG gating strategy. Two- or three color PLG protocol, in double platform, currently seems the most accurate and affordable method to monitor CD4+ lymphocytes and CD4/CD8 ratio by flow cytometry in resource-poor medical settings.     

Comparative phylogeography of Zacco platypus and Opsariichthys bidens (Teleostei, Cyprinidae) in China based on cytochrome b sequences

We compared the phylogenetic relationships and the population structure of the sympatric cyprinids Opsariichthys bidens and Zacco platypus across the Yangzte, Pearl and Hai He drainages in Mainland China. We used the mitochondrial cytochrome b gene to evaluate their phylogeographical structure and to discuss further systematic implications. We sequenced 71 new individuals of Z. platypus to add to a previous study and to construct a geographically comparable data set with O. bidens (Mol. Phylogenet. Evol. 31, 2004, 192; Mol. Phylogenet. Evol. 37, 2005, 920). Phylogeographic analysis of Z. platypus identified two additional mtDNA lineages (Zacco E and F) in addition to the previously recovered lineages (Zacco A–D). The geographical range of the Zacco A was extended across the Pearl drainage, broadening considerably the previous distribution that was circumscribed to the Yangtze drainage. Our results show high molecular divergence between all Zacco A–F mtDNA lineages (mean TrN + I + G distance 17.7%, range 4.4–30.8%), and the population analyses showed that most of the genetic variation was found among major river tributaries (90.2%). Values of θ_ST higher than 0.97 corroborated this interruption of gene flow between Zacco A–F mtDNA lineages. Nested clade analysis results were also concordant with the phylogenetic relationships recovered. We found similar phylogeographic patterns in Z. platypus and O. bidens: they exhibited two widespread and common lineages (Zacco A and Opsariichthys 1) ranging across the Yangtze and Pearl drainages, and both taxa had Northern lineages (Zacco F and Opsariichthys 5) clearly differentiated. Similar phylogeographic patterns suggest that common historical factors have shaped current Zacco and Opsariichthys diversity. High genetic distances and θ_ST values suggested that differentiation of Zacco A–F mtDNA lineages and Opsariichthys 1–5 was not recent and was probably favoured by old geological events. However, population parameters and low genetic differentiation inside Zacco A and Opsariichthys 1 suggest a recent gene flow across river drainages. Mitochondrial‐based phylogenies for the combined data sets of Zacco A–F and Opsariichthys 1–5 mtDNA lineages always recovered both taxa as paraphyletic, highlighting the notion that their current systematics need a revision.     

MMP25 (MT6-MMP) is highly expressed in human colon cancer, promotes tumor growth, and exhibits unique biochemical properties

MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (K(i) = 0.2 nm) over TIMP-2 (K(i) = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface.     

The chemokine CCL6 promotes innate immunity via immune cell activation and recruitment

Septic syndrome is a consequence of innate immune failure. Recent studies showed that the CC chemokine CCL6 enhanced antimicrobial immunity during experimental sepsis through an unknown mechanism. The present study demonstrates that transgenic CCL6 expression abolishes mortality in a septic peritonitis model via the modulation of resident peritoneal cell activation and, more importantly, through the recruitment of IFN-producing NK cells and killer dendritic cells into the peritoneum. Thus, CCL6 attenuates the immune failure during sepsis, in part, through a protective type 1-cytokine mediated mechanism.     

Antioxidant activity of leaf extracts from Bauhinia monandra

Bauhinia monandra Kurz. is used in Brazil for the treatment of diabetes. Since this activity may be correlated with the presence of antioxidant compounds, leaf extracts of B. monandra were evaluated for their radical scavenging capacity (RSC). An ethanolic extract was taken up in aqueous methanol and partitioned with hexane, chloroform, ethyl acetate to yield three organic extracts together with remaining aqueous extract. The RSC was determined spectrophotometrically using 1,1-diphenylpicrylhydrazyl free radical (DPPH). The chloroform and ethyl acetate extracts were the most appropriate as sources of antioxidant compounds as shown by their inhibition concentration (IC50) and inhibition percentage (IP) values. The antioxidant activity of such extracts was attributed to the presence of three compounds of different polarities (flavonoids and steroids). The chloroform and ethyl acetate extracts exhibited an IC50 of approximately 2 mg/g DPPH and IP values in the range of 60-65%. The results indicate that the extracts of B. monandra have a very potent antioxidant activity, compared with the pure catechins used as positive controls and with other plant extracts.     

Long-term stability studies on protection against Newcastle disease by commercial live vaccine used in Brazil

The thermostability (TS) and efficacy offered by live vaccines against Newcastle disease strains B1, La Sota, VG-GA and Ulster, produced or imported by four Brazilian laboratories, were evaluated during their validity period. Kinetic profiles were obtained from samples conserved in refrigerators during 0, 4, 8, 12, 16, 20 and 24 months after their manufacturing. The statistical analysis of the vaccine titre effect obtained by the fresh air (FA) method showed that the vaccine profiles were parallel and coincident, presenting a significant descending trend. The vaccine titres and efficiency proofs at the end of the validity period were above the level of legislation requirements and showed an average loss in titre of 0.40 and 0.66 log_10, within the first and second validity years, respectively. The titre obtained by TS, within the month after manufacturing, had no significant difference from the titre obtained by FA within 24 months after manufacturing, being their pairs of observations positively correlated (r = 0.49, p = 0.0003), showing that the TS method, which anticipates the vaccines' performance at the end of the validity period, can substitute the FA method 24 months after manufacturing.     

Inosine triphosphatase allele frequency and association with ribavirin-induced anaemia in Brazilian patients receiving antiviral therapy for chronic hepatitis C

Inosine triphosphatase (ITPA) single nucleotide polymorphisms (SNPs) are stronglyassociated with protection against ribavirin (RBV)-induced anaemia in European,American and Asian patients; however, there is a paucity of data for Brazilianpatients. The aim of this study was to evaluate the ITPA SNP (rs7270101/rs1127354)frequency in healthy and hepatitis C virus (HCV)-infected patients from Brazil andthe association with the development of severe anaemia during antiviral therapy. ITPASNPs were determined in 200 HCV infected patients and 100 healthy individuals bysequencing. Biochemical parameters and haemoglobin (Hb) levels were analysed in 97patients who underwent antiviral therapy. A combination of AArs7270101+CCrs1127354(100% ITPase activity) was observed in 236/300 individuals. Anaemia was observed in87.5% and 86.2% of treated patients with AA (rs7270101) and CC genotypes (rs1127354),respectively. Men with AA (rs7270101) showed a considerable reduction in Hb at week12 compared to those with AC/CC (p = 0.1475). In women, there was no influence ofgenotype (p = 0.5295). For rs1127354, men with the CC genotype also showed a suddenreduction in Hb compared to those with AC. Allelic distribution of rs7270101 andrs1127354 shows high rates of the genotypes AA and CC, respectively, suggesting thatthe study population had a great propensity for developing RBV-induced anaemia. Aprogressive Hb reduction during treatment was observed; however, this reduction wasgreater in men at week 12 than in women.     

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.