Equal Numbers of Neuronal and Nonneuronal Cells Make the Human Brain an Isometrically Scaled-Up Primate Brain

The human brain is often considered to be the most cognitively capable among mammalian brains and to be much larger than expected for a mammal of our body size. Although the number of neurons is generally assumed to be a determinant of computational power, and despite the widespread quotes that the human brain contains 100 billion neurons and ten times more glial cells, the absolute number of neurons and glial cells in the human brain remains unknown. Here we determine these numbers by using the isotropic fractionator and compare them with the expected values for a human-sized primate. We find that the adult male human brain contains on average 86.1 +/- 8.1 billion NeuN-positive cells (""neurons"") and 84.6 +/- 9.8 billion NeuN-negative (""nonneuronal"") cells. With only 19% of all neurons located in the cerebral cortex, greater cortical size (representing 82% of total brain mass) in humans compared with other primates does not reflect an increased relative number of cortical neurons. The ratios between glial cells and neurons in the human brain structures are similar to those found in other primates, and their numbers of cells match those expected for a primate of human proportions. These findings challenge the common view that humans stand out from other primates in their brain composition and indicate that, with regard to numbers of neuronal and nonneuronal cells, the human brain is an isometrically scaled-up primate brain. J. Comp. Neurol. 513:532-541, 2009. (c) 2009 Wiley-Liss, Inc.

Frequency of human metapneumovirus infection in hematopoietic SCT recipients during 3 consecutive years

Respiratory viruses can cause significant morbidity in immunocompromised hosts. Human metapneumovirus (hMPV) has been increasingly associated with lower respiratory tract infection in hematopoietic SCT (HSCT) recipients, with mortality rates up to 50%. No data on the occurrence of hMPV infection in HSCT recipients have been reported in the southern hemisphere. We conducted a retrospective study including 228 nasal wash samples from 153 HSCT recipients with respiratory symptoms during 2001, 2002 and 2003. hMPV was detected by real-time PCR with primers complementary to the nucleocapsid region of hMPV genome. Eleven of the 153 patients (7.2%) acquired hMPV infection during the study period (6.4% in 2001, 4.7% in 2002 and 11.1% in 2003). Among the 11 HSCT recipients with hMPV infection, 1 died 8 days after the diagnosis, but the role of hMPV in the patient`s death could not be established. In 2001 and 2003, hMPV group A prevailed over group B. In 2002, both groups were detected equally. hMPV infections were diagnosed in late winter and spring. The frequency of hMPV infection in HSCT recipients living in Brazil was similar to those observed in the northern hemisphere. Sensitive techniques to detect hMPV should be included in the diagnostic assessment of HSCT recipients with respiratory symptoms.

High numbers of human skin cancers express MMP2 and several integrin genes

Background: Basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and cutaneous malignant melanoma (MM) are solid skin cancers derived from different cell types, with different ability to metastasize. Several subtypes of integrins and matrix metalloproteinases (MMP) have been related to malignization and metastasis processes. This work aimed at a quantitative evaluation of skin cancers expressing eight integrins and MMP2 genes. Methods: Expression of integrins and MMP2 genes was evaluated on fresh tumor biopsies from BCC, SCC and MM, and respective controls, by the reverse transcriptase polychain reaction (RT-PCR) technique. Results: More than 90% tumors expressed alpha 6a, beta 1, beta 3 and beta 6 (non-melanoma), and alpha 5a, alpha 6a and MMP2 (MM). Up to 100% controls also expressed beta 1 and beta 3. The results were significant for alpha 6a in BCC (p = 0.026), alpha 6b in SCC (p = 0.035), alpha 2a in BCC (p = 0.003), beta 5 and beta 6 in BCC (p = 0.005). MMP2 was expressed in 100% MM (p = 0.0004). Conclusion: Integrin subunits alpha 2a and alpha 6a would be interesting targets for BCC anti-tumor therapy, as well as alpha 6b in case of SCC. The elevated number of BCC expressing alpha 2 and alpha 6, and of MM expressing alpha v and MMP2, corroborate literature data.

Toxoplasma gondii isolates: Multi locus RFLP-PCR genotyping from human patients in Sao Paulo State, Brazil identified distinct genotypes

This study investigated the genetic characteristics of Toxoplasma gondii samples collected from 62 patients with toxoplasmosis in Sao Paulo State, Brazil. DNA samples were isolated from blood, cerebrospinal fluid and amniotic fluids of 25 patients with cerebral toxoplasmosis and AIDS, two patients with acute toxoplasmosis, 12 patients with ocular toxoplasmosis, six newborns with congenital toxoplasmosis and 17 pregnant women with acute infection. Diagnosis of toxoplasmosis was based in clinical, radiological and laboratory features. Genotyping was performed using multilocus PCR-RFLP genetic markers including SAG1, SAG2, 5`- and 3`-SAG2, alt.SAG2, SAG3, BTUB, GRA6, C22-8, c29-2, L358, PK1 and Apico. Among the 62 clinical samples, 20 (32%) were successfully genotyped at eight or more genetic loci and were grouped to three distinct genotypes. Eighteen samples belonged to ToxoDB Genotype #65 and the other two samples were identified as ToxoDB Genotypes #6 and #71, respectively (http://toxodb.org/toxo/). Patients presenting Genotypes #6 and #71 had severe and atypical cerebral toxoplasmosis, characterized by diffuse encephalitis without extensive brain lesions. These results indicate that T. gondii Genotype #65 may have a high frequency in causing human toxoplasmosis in Sao Paulo State, Brazil. This unusual finding highlights the need to investigate the possible association of parasite genotypes with human toxoplasmosis. (C) 2011 Elsevier Inc. All rights reserved.

PHOTOELASTIC ANALISYS OF A HUMAN VERTEBRA MODEL WITH PEDICULAR SCREW

Introduction: The vertebrae fixation system using pedicular screws is one of the most efficient methods to treat vertebral spine pathologies. When the screw is submitted to pullout strength, it causes internal tension near the medullar canal and this situation can be analyzed by using the photoelasticity technique. Objective: Were analyzed those internal tensions near the medullar canal of photoelastic vertebra models using different sizes of screws of the vertebral fixation system submitted to pullout strength. Methods: A lumbar vertebral model made of photoelastic material with three different USS1-type pedicular screw sizes (5, 6, and 7 mm) was used. The internal tensions around the screw were tested in 12 predetermined points by a plain transmission polaroscope. Results: The areas of greater tension concentration were between the medullar canal and the curves of the transverse process. Comparing the maximum average pulling tension, statistical differences were observed between screws 5 and 7, and 6 and 7. On the other hand, for screws 5 and 6, there were no significant differences. Conclusion: The study evidenced that the internal tensions are greater in irregular areas, next to the medullar canal, showing that this is a critical region.

The ""single-section"" Golgi method adapted for formalin-fixed human brain and light microscopy

The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The ""single-section"" Golgi method of Gabbott and Somogyi (1984) and the modifications made by Izzo et al. (1987) are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, ""sandwiched"" between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the ""single-section"" Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results. (C) 2010 Elsevier B.V. All rights reserved.

FREQUENCY OF HUMAN RHINOVIRUS SPECIES IN OUTPATIENT CHILDREN WITH ACUTE RESPIRATORY INFECTIONS AT PRIMARY CARE LEVEL IN BRAZIL

This study assessed the occurrence of human rhinovirus (HRV) species in outpatient children attending day-care in Sao Paulo, Brazil. HRV reverse transcriptase polymerase chain reaction and amplicon sequencing were done in 120 samples collected in 2008. HRV was detected in 27.5% of samples. HRV C was detected in 60.7% of wheezers, a frequency not different from that observed in nonwheezers (69.6%).

Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children. (C) 2007 Wiley-Liss, Inc.

Stable and high-level production of recombinant Factor IX in human hepatic cell line

Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries.

Hepatitis B viral markers in banked human milk before and after Holder pasteurization

Background: Blood screening for hepatitis B virus (HBV) is not universally performed for donor selection in human milk banks. Objectives: To evaluate the frequency of detection of HBV surface antigen (HBsAg) and HBV-DNA in colostrum of HBV-infected nursing mothers before and after Holder pasteurization. Study design: Forty-two concentrated breast milk samples were obtained within two postnatal weeks from 24 HBsAg-positive women (4 HBeAg-positive and 20 HBeAg-negative, anti-HBe-positive) were tested for the presence of HBsAg and HBV-DNA before and after Holder pasteurization (30 min at 62.5 degrees C). Results: Before pasteurization, HBsAg and HBV-DNA were found in 14/24 (58%), and 20/24 (75%) first milk samples, respectively, obtained by 4 days after delivery. At least one marker was detected in 20/24 (83%) milk samples. Both markers were identified in milk of HBeAg-positive mothers, and most mothers with anti-HBe in blood had at least one HBV marker. Once detected, viral markers were frequently found in milk samples subsequently obtained from the same woman. Holder pasteurization did not affect the probability of detecting HBsAg (8/18, 44%), HBV-DNA (12/18, 67%). or at least one of them (15118, 83%). Conclusions: Although the biological implications of these findings remain to be determined, considering that HBV is highly contagious and most recipients of banked human milk are preterm infants, these findings should be taken into account when donors are enlisted for human milk banks without serological screening. (C) 2009 Elsevier B.V. All rights reserved.

HLA-G polymorphisms in women with squamous intraepithelial lesions harboring human papillomavirus

Human papillomavirus (HPV) infection is etiologically associated with low-(LSIL) and high-grade squamous intraepithelial lesions (HSIL) and with cervical cancer. The progression or regression of the lesions may depend, among other factors, on the host heritable immune response. Because human leukocyte antigen (HLA)-G molecules are involved in the modulation of innate and adaptive immune responses, and because no previous studies have evaluated HLA-G polymorphism in patients with SIL, we conducted a study to assess the association between HLA-G polymorphisms and cervical lesions harboring HPV infection. Cervico-vaginal scrapings and blood samples were collected from 125 women with SIL (68 LSIL and 57 HSIL) and from 94 healthy women without HPV infection and cytological abnormalities. HPV type and HLA-G polymorphisms in exons 2, 3 and 8 (14 bp insertion/deletion) were evaluated by PCR methodology, and digested with restriction endonucleases. The Genepop software and the EM and PHASE algorithms were used for statistical analysis. A significant protective association was observed between the presence of the G*0103 allele and SIL and between the G0101/G0104 genotype and HSIL in the group of patients compared to control. The presence of the G0104/+14 bp and G0104/-14 bp haplotypes conferred susceptibility to SIL compared to control. In addition, patients possessing the G0104/+14 bp haplotype and harboring HPV-16 and -18 co-infections were particularly associated with HSIL. These findings suggest that HLA-G polymorphisms may be associated with HPV infection and SIL, consequently representing a profile of predisposition to cervical cancer. Modern Pathology (2009) 22, 1075-1082; doi: 10.1038/modpathol.2009.67; published online 1 May 2009

Histoplasma capsulatum Cell Wall beta-Glucan Induces Lipid Body Formation through CD18, TLR2, and Dectin-1 Receptors: Correlation with Leukotriene B(4) Generation and Role in HIV-1 Infection

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of worldwide significance. Infection with Hc produces a broad spectrum of diseases and may progress to a life-threatening systemic disease, particularly in individuals with HIV infection. Resolution of histoplasmosis is associated with the activation of cell-mediated immunity, and leukotriene B(4) plays an important role in this event. Lipid bodies (LBs) are increasingly being recognized as multifunctional organelles with roles in inflammation and infection. In this study, we investigated LB formation in histoplasmosis and its putative function in innate immunity. LB formation in leukocytes harvested from Hc-infected C57BL/6 mice peaks on day 2 postinfection and correlates with enhanced generation of lipid mediators, including leukotriene B(4) and PGE(2). Pretreatment of leukocytes with platelet-activating factor and BLT1 receptor antagonists showed that both lipid mediators are involved in cell signaling for LB formation. Alveolar leukocytes cultured with live or dead Hc also presented an increase in LB numbers. The yeast alkali-insoluble fraction 1, which contains mainly beta-glucan isolated from the Hc cell wall, induced a dose- and time-dependent increase in LB numbers, indicating that beta-glucan plays a signaling role in LB formation. In agreement with this hypothesis, beta-glucan-elicited LB formation was inhibited in leukocytes from 5-LO(-/-), CD18(low) and TLR2(-/-) mice, as well as in leukocytes pretreated with anti-Dectin-1 Ab. Interestingly, human monocytes from HIV-1-infected patients failed to produce LBs after beta-glucan stimulation. These results demonstrate that Hc induces LB formation, an event correlated with eicosanoid production, and suggest a role for these lipid-enriched organelles in host defense during fungal infection. The Journal of Immunology, 2009, 182: 4025-4035.

CXCL12 rs1801157 Polymorphism in Patients with Breast Cancer, Hodgkin`s Lymphoma, and Non-Hodgkin`s Lymphoma

Chemokines and their receptors regulate the trafficking of immune cells during their development, inflammation, and tissue repair. The single-nucleotide polymorphism (SNP) rs1801157 (previously known as CXCL12-A/ stromal cell-derived factor-1 (SDF1)-3`A) in CXCL12/SDF1 gene was assessed in breast cancer, Hodgkin`s lymphoma (HL), and non-Hodgkin`s lymphoma (NHL), since the chemokine CXCL12, previously known as SDF1, and its receptor CXCR4 regulate leukocyte trafficking and many essential biological processes, including tumor growth, angiogenesis, and metastasis of different types of tumors. Genotyping was performed by PCR-RFLP (polymerase chain reaction followed by restriction fragment length polymorphism) using a restriction enzyme Hpall cleavage. No significant difference was observed in genotype distribution between breast cancer patients (GG: 57.3%; GA: 39.8%; AA: 2.9%) and healthy female controls (GG: 62.9%; GA: 33%; AA: 4.1%) nor between HL patients (GG: 61.1%; GA:27.8%; AA: 11.1%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%), whereas a significant difference was observed in genotype distribution between NHL patients (GG: 51.4%; GA: 47.1%; AA: 1.5%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%). Further studies will be necessary to elucidate the cancer chemokine network. However, this study suggests that CXCL12 rs1801157 polymorphism may have important implications in the pathogenesis of NHL. J. Clin. Lab. Anal. 23:387-393, 2009. (C) 2009 Wiley-Liss, Inc.

Investigation of the in vivo activity of CYP3A in Brazilian volunteers: comparison of midazolam and omeprazole as drug markers

Objective This study compares midazolam with omeprazole as marker drugs for the evaluation of CYP3A activity in nine healthy self-reported white Brazilian volunteers. Methods Omeprazole was also used to evaluate the CYP2C19 phenotype. The volunteers received p.o. 20 mg omeprazole, and blood samples were collected 3.5 h after drug administration. After a washout period of 10 days, the volunteers received p.o. 15 mg midazolam maleate, and serial blood samples were collected up to 6 h after administration of the drug. CYP2C19 was genotyped for the allelic variants CYP2C19*1, CYP2C19*2, CYP2C19*3, and CYP2C19*17. Analysis of omeprazole, hydroxyomeprazole, omeprazole sulfone, and midazolam in plasma was carried out by LC-MS/MS. Results The volunteers genotyped as CYP2C19*1*17, CYP2C19*17*17, CYP2C19*1*1 (n=8), or CYP2C19*17*2 (n=1) presented a median hydroxylation index (omeprazole/hydroxyomeprazole) of 1.35, indicating that all of them were extensive metabolizers of CYP2C19. The volunteers (n=9) presented a 0.12 log of the omeprazole/sulfone ratio and a median oral clearance of midazolam of 17.89 ml min(-1) kg(-1), suggesting normal CYP3A activity. Conclusions Orthogonal regression analysis between midazolam clearance and log of the plasma concentrations of the omeprazole/omeprazole sulfone ratio (R=-0.7544, P < 0.05) suggests that both midazolam and omeprazole can be used as markers of CYP3A activity in the population investigated.

Effect of a thrombin receptor (protease-activated receptor 1, PAR-1) gene polymorphism in chronic hepatitis C liver fibrosis

Background and Aim: Tissue injury leads to activation of coagulation and generation of thrombin. Inhibition of thrombin receptor protease-activated receptor 1 (PAR-1) has been shown to reduce liver fibrosis in animals. This study aimed to evaluate the effect of PAR-1 gene polymorphism on rate of liver fibrosis (RF) in chronic hepatitis C. Methods: Polymorphisms studied: C > T transition 1426 bp upstream of translation start site (-1426C/T), 13 bp repeat of preceding -506 5`-CGGCCGCGGGAAG-3` sequence (-506I/D), and A > T transversion in intervening sequence (IVS) 14 bp upstream of exon-2 start site (IVS-14A/T). A total of 287 European and 90 Brazilian patients were studied. Results: 1426C/T polymorphism: There was a trend to higher RF in patients with the TT genotype (P = 0.06) and an association between genotype CC and slow fibrosis (P = 0.03) in Europeans. In males, RF was significantly higher in those with the TT genotype compared to CT (P = 0.003) and CC (P = 0.007). There was a significant association between TT and fast fibrosis (P = 0.04). This was confirmed in an independent cohort of Brazilians where RF was higher in TT than in CC (P = 0.03). Analysis of -506I/D showed no difference in RF and distribution of slow/fast fibrosis among different genotypes in both populations. Analysis of IVS-14A/T showed no difference between genotypes. Conclusion: In conclusion, these findings suggest that PAR-1 receptor polymorphisms influence the progression of liver fibrosis.