Association study of candidate DNA-repair gene variants and acute graft versus host disease in pediatric patients receiving allogeneic hematopoietic stem-cell transplantation.

Acute Graft versus Host Disease (aGvHD) grades 2-4 occurs in 15-60% of pediatric patients undergoing allogeneic haematopoietic stem-cell transplantation (allo-HSCT). The collateral damage to normal tissue by conditioning regimens administered prior to allo-HSCT serve as an initial trigger for aGvHD. DNA-repair mechanisms may play an important role in mitigating this initial damage, and so the variants in corresponding DNA-repair protein-coding genes via affecting their quantity and/or function. We explored 51 variants within 17 DNA-repair genes for their association with aGvHD grades 2-4 in 60 pediatric patients. The cumulative incidence of aGvHD 2-4 was 12% (n = 7) in the exploratory cohort. MGMT rs10764881 (G>A) and EXO rs9350 (c.2270C>T) variants were associated with aGvHD 2-4 [Odds ratios = 14.8 (0 events out of 40 in rs10764881 GG group) and 11.5 (95% CI: 2.3-191.8), respectively, multiple testing corrected p ≤ 0.001]. Upon evaluation in an extended cohort (n = 182) with an incidence of aGvHD 2-4 of 22% (n = 40), only MGMT rs10764881 (G>A) remained significant (adjusted HR = 2.05 [95% CI: 1.06-3.94]; p = 0.03) in the presence of other clinical risk factors. Higher MGMT expression was seen in GG carriers for rs10764881 and was associated with higher IC50 of Busulfan in lymphoblastoid cells. MGMT rs10764881 carrier status could predict aGvHD occurrence in pediatric patients undergoing allo-HSCT.

Association of CTH variant with sinusoidal obstruction syndrome in children receiving intravenous busulfan and cyclophosphamide before hematopoietic stem cell transplantation.

Sinusoidal obstruction syndrome (SOS) is a severe complication of hematopoietic stem cell transplantation (HSCT) that can be fatal, often attributed to the conditioning regimen prior to HSCT. We evaluated the association of SOS risk with gene variants in cystathionase (CTH), an enzyme involved in glutathione synthesis, in 76 children receiving intravenous busulfan (Bu) before HSCT. Our results indicated an association with CTHc.1364 G>T (ORTT=10.6, 95% confidence interval (CI)=2.16, 51.54) and SOS risk, which was sex dependent (female patients, ORTT=21.82, 95% CI=3.590-132.649). The interaction between CTHc.1364 G>T and another risk variant (GSTA1*B) was explored. A recessive model with the use of GSTA1*B*B and CTH c.1364 TT genotypes proved to be useful at predicting SOS occurrence, indicating the possibility of using these gene variants as markers of SOS occurrence and to further individualize preemptive treatment aimed at reducing SOS incidence.

The association of cytochrome P450 genetic polymorphisms with sulfolane formation and the efficacy of a busulfan-based conditioning regimen in pediatric patients undergoing hematopoietic stem cell transplantation.

Cytochrome P450 enzymes (CYPs) and flavin-containing monooxygenases (FMOs) likely have a role in the oxidation of intermediate metabolites of busulfan (Bu). In vitro studies to investigate the involvement of these enzymes are cumbersome because of the volatile nature of the intermediate metabolite tetrahydrothiophene (THT) and the lack of sensitive quantitation methods. This study explored the association between the CYP2C9, CYP2C19, CYP2B6 and FMO3 genotypes and sulfolane (Su, a water soluble metabolite of Bu) plasma levels in children undergoing hematopoietic stem cell transplantation (HSCT). The relationship between these genotypes and the effectiveness of myeloablative conditioning was also analyzed. Sixty-six children receiving an intravenous Bu-based myeloablative conditioning regimen were genotyped for common functional variant alleles in CYP2C9 (*2 and *3), CYP2C19 (*2 and *17), FMO3 (rs2266780, rs2266782 and rs1736557) and CYP2B6 (*5 and *9). The plasma levels of Bu and its metabolite Su were measured after the ninth Bu dose in a subset of 44 patients for whom plasma samples were available. The ratio of Bu to Su was considered the metabolic ratio (MR) and was compared across the genotype groups. Higher MRs were observed in CYP2C9*2 and *3 allele carriers (mean±s.d.: 7.8±3.6 in carriers vs 4.4±2.2 in non-carriers; P=0.003). An increased incidence of graft failure was observed among patients with an MR>5 compared with those with MR values <5 (20% vs 0%; P=0.02). In contrast, a significantly higher incidence of relapse and graft failure (evaluated as event-free survival) was observed in patients with malignant disease who carried CYP2B6 alleles with reduced function on both chromosomes compared with carriers of at least one normal allele (100% vs 40%; P=0.0001). These results suggest that CYP2C9 has a role in the oxidation reactions of THT and indicate that it may be possible to predict the efficacy of Bu-based myeloablative conditioning before HSCT on the basis of CYP genotypes and Bu MRs.

Short-term Results of the Masking Effect of an Infiltrant Resin on Mild Molar Incisor Hypomineralization Lesions in Anterior Teeth.

This non-controlled clinical study evaluated the masking effect of an infiltrant resin on mild molar incisor hypomineralization (MIH) lesions. Thirty MIH-affected anterior teeth with creamy/ white opacities from 12 children aged 6-15 years received the application of an infiltrant resin (Icon- DMG). Standard photographs were taken before (T1), immediately after (T2), and 1 week after (T3) resin infiltration. Two calibrated examiners qualitatively analyzed the color match using the Fédération Dentaire Internationale (FDI) scale. The binomial distribution test analyzed the scores of the color match at T1 with T2 and T3, and McNemar's test analyzed the scores of the color match between T2 and T3 (α=0.05). There was a significant increase in color match between T1 and T2 (p=0.0005), between T1 and T3 (p=0.0005), and between T2 and T3 (p=0.0019). It was concluded that infiltrant resin was effective in improving the esthetic appearance of creamy/white opacities on MIH-affected anterior teeth.

Saliva and blood interferon gamma levels and IFNG genotypes in acute graft-versus-host disease.

OBJECTIVE: Graft-versus-host disease is a major complication after allogenic hematopoietic stem cell transplantation. Interferon gamma is an important pro-inflammatory cytokine involved in this disease. Cytokine gene polymorphisms are associated with functional differences in cytokine expression and can alter the clinical course of graft-versus-host disease. This study aimed to investigate the association between IFN-γ levels in saliva, blood, and IFNG polymorphisms, as well as the occurrence of acute graft-versus-host disease in allogenic HSCT. SUBJECTS AND METHODS: Fifty-eight consecutive allogenic hematopoietic stem cell transplantation recipients and their donors were prospectively studied. IFN-g levels in saliva and blood were assessed by ELISA. Samples were collected weekly from 7 days before transplantation (day -7) to 100 days after allogenic HSCT (day +100) or until death. Saliva and/or blood samples were obtained from the recipients and donors to determine IFNG gene polymorphisms. RESULTS: Increased saliva and blood IFN-g levels were observed in patients that had developed aGVHD. In the saliva, the peak levels of IFN-g could be found one week before aGVHD diagnosis, while in the blood, peak levels of IFN-g could be only observed upon diagnosis. A significant association could be identified between the recipients'IFNG genotypes and the IFN-g levels in their blood, at +14 days after HSCT. No association could be observed between IFNG gene polymorphisms and the aGVHD. CONCLUSION: The present study shows that the genetic background of recipients can influence the production of IFN-g. Moreover, as IFN-g levels in the saliva and blood were found to be associated with aGVHD development, this cytokine may be a useful predictor of acute graft-versus-host disease.

Retention of Manually or CAD/CAM-customized Fiberglass Posts Luted to Enlarged Root Canals with Different Resin Cements.

The aim of this laboratory study was to evaluate the pull-out force of a prefabricated fiberglass post (PP), relined fiberglass post (RP), or milled fiberglass post (MP) luted with Multilink N (MN), RelyX Unicem 2 (RXU2) or RelyX Ultimate (RU) to enlarged root canals. The thickness of the resin cements and the presence of voids in the resin cement film were observed. The root canals of 90 bovine incisors were enlarged, endodontically treated, and randomly divided into 9 groups (n=10) according to the post type and resin cement. The specimens were scanned using micro-CT to analyze the thickness of the resin cement and the presence of voids. The specimens were submitted to mechanical cyclic loading (500,000 cycles at 50 N load) and subjected to pull-out force testing. Two-way ANOVA and Tukey's test analyzed the pull-out force and resin cement thickness data. Kruskal-Wallis and Bonferroni tests analyzed the void scores. The interaction between factors (post × resin cement) was significant (p=0.0001) for the pull-out force. Higher pull-out forces were obtained for RP and MP compared to PP. The post factor was significant (p=0.0001) for resin cement thickness, which was higher for PP (1054 µm), followed by MP (301 µm) and RP (194 µm). More void formation occurred for PP, being less for RP, differing significantly among the posts. Post customization (RP and MP) decreased resin cement thickness and void formation, favoring a higher pull-out force. Resin cements requiring an adhesive application (MN and RU) favored higher pull-out force than self-adhesive resin cement (RXU2).

HCMV gB genotype and its association with cytokine levels in hematopoietic stem cell transplantation.

BACKGROUND: Glycoprotein B (gB) has been implicated in determining the pathogenicity and clinical outcomes of human cytomegalovirus (HCMV) disease. OBJECTIVE: The purpose of this study was to assess the prevalence of gB genotypes in allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the relationship between it and cytokine levels in saliva and blood samples. The impact of these parameters on patients' survival was also investigated. METHODS: Samples were obtained from 63 patients receiving an allo-HSCT. HCMV gB genotyping was carried out by multiplex nested PCR. The cytokine levels were assessed using ELISA assay. RESULTS: A single or mixed genotype infection was detected in the saliva and blood of 36/63 and 52/63 subjects, respectively. Patients with gB2 in their saliva showed lower IL-10 levels in comparison with patients without gB2. Reduced blood levels of IFN-γ and IL-1ß were also found in recipients with the HCMV gB4 genotype compared with patients without it. Decreased IL-1ß and increased IL-10 blood levels were associated with lower survival. However, HCMV gB genotypes have no impact on patient outcome. CONCLUSION: Decreased IL-1ß and increased IL-10 levels in the blood are associated with lower survival. HCMV genotypes are associated with different cytokine levels in saliva and blood.

Saliva as a source of HCMV DNA in allogeneic stem cell transplantation patients.

OBJECTIVE: The purpose of this study was to investigate the use of saliva for the identification of human cytomegalovirus (HCMV) in allogeneic hematopoietic stem cell transplant patients by real time PCR compared with blood. MATERIALS AND METHODS: Saliva and blood samples were sampled weekly in 30 allogeneic hematopoietic stem cell transplant patients until 100 days after transplant. Total genomic DNA, extracted from saliva and whole-blood samples, was used for HCMV real time PCR. Nonparametric tests were performed, and P value

Effect of pH Cycling Followed by Simulated Toothbrushing on the Surface Roughness and Bacterial Adhesion of Bulk-fill Composite Resins.

The aim was to evaluate, in vitro, quantitatively and qualitatively, the effect of pH cycling and simulated toothbrushing on surface roughness (Ra) and bacterial adhesion (Cn) of bulk-fill composite resins. Thirty specimens of each composite resin, 5 mm wide and 4 mm high, were obtained: group 1 (control): Filtek Z250 (Z250); group 2: Filtek Bulk-Fill (FTK); group 3: Tetric N-Ceram Bulk-Fill (TTC); and group 4: Aura Bulk-Fill (AUR). After 24 hours, the specimens were polished and then alternated with demineralization/remineralization solutions for 15 cycles of 24 hours each at 37°C. Then the specimens were submitted to simulated toothbrushing. The Ra and Cn measurements were quantitatively analyzed in three stages: after polishing (Ra0 and Cn0), after pH cycling (Ra1 and Cn1), and after simulated toothbrushing (Ra2 and Cn2). The Ra values were submitted to two-way analysis of variance, followed by the Tukey test (α=0.05). The Kruskal-Wallis test, followed by multiple comparisons, was applied for Cn analysis. Surface topography and bacterial adhesion were observed by scanning electron microscopy (SEM). Z250, FTK, and TTC showed no significant change in Ra regardless of the treatment performed; AUR obtained increased Ra at Ra2 (p<0.05). FTK differed from the others at Cn0 and Cn1 (p<0.05). At Cn2, there was no difference among the composite resins. SEM images showed the exposure of fillers and microcavities at Ra1 and Ra2. There was greater bacterial adhesion at Cn1 for Z250 and FTK. It was concluded that the pH cycling caused surface degradation of all composite resins, which was potentiated by simulated toothbrushing. However, only AUR presented an increased Ra. Bacterial adhesion occurred on all composite resins after pH cycling; however, after simulated toothbrushing, adhesion of dispersed bacteria was similar for all the composite resin groups.

Human pegivirus-1 (HPgV-1) RNA prevalence and genotypes in volunteer blood donors from the Brazilian Amazon.

OBJECTIVES: The objectives of this study were to evaluate the prevalence of Human Pegivirus-1 (HPgV-1) viremia and genotype diversity among healthy blood donors from the Eastern Brazilian Amazon (city of Macapá, State of Amapá). There is little information for prevalence and circulation of HPgV-1 in this remote Brazilian region. MATERIALS AND METHODS: We conducted a study evaluating the HPgV-1 RNA prevalence and circulating genotypes in 431 volunteer blood donors originating from the Eastern Brazilian Amazon. The obtained HPgV-1 positive samples were submitted to sequencing and genotyping analysis in order to examine the genotype diversity of this virus in the Brazilian Amazon. RESULTS: Our results demonstrated a prevalence of HPgV-1 RNA in 9.5% of the tested blood donors. The phylogenetic analyses of the detected positive samples showed the presence of HPgV-1 genotypes 1, 2 and 3. The most frequently detected genotype was 2 (78.0% of the cases) represented by sub-genotypes 2A (39.0%) and 2B (39.0%). At lower rates, genotypes 1 (14.6%) and 3 (7.4%) were also detected. CONCLUSION: Our results revealed the presence of genotypes with European, Asiatic and African endemicity in Amazonian blood donors, probably due to the complex miscegenation processes that took place in this Brazilian region. More investigations, including information for the prevalence of HPgV-1 RNA in blood donors from other Latin American countries are needed to estimate the viremic rates and genotype distribution of this virus in a highly diverse continent like South America.

Efficacy of glutamine-supplemented parenteral nutrition on short-term survival following allo-SCT: a randomized study.

Fifty-three patients with hematological malignancies who underwent Allo-SCT from HLA-identical siblings were randomly assigned to receive glutamine-enriched parenteral nutrition-PN (GlPN, n=27) or standard PN (PN, n=26), in isonitrogenous solutions. Deaths (D+100 and D+180), infections, acute GVHD, length of stay, time of neutropenia and intestinal permeability (IP) were studied. Ages, gender, diagnosis, disease status and treatment variables were equally distributed between groups. Survival on D+180 was increased in GlPN (74%) vs PN (46%), P=0.03 (log-rank), as on D+100 (P=0.05). Most deaths occurred before D+100, especially in PN (10/26, 39%) vs GlPN (4/27, 15%). GVHD was the most frequent cause of death (8/21, 38%), especially in PN (n=6, five before D+100). Other outcomes were not affected. IP was affected on admission, was not affected by glutamine enrichment, but consistently worsened throughout the study. Results showed that GlPN was efficacious in increasing short-term survival after Allo-SCT. Benefits of glutamine seem to be independent of mucosal protection, as IP was not affected by its use. A trend to a lower incidence of GVHD deaths may suggest an immunomodulatory role of glutamine.

Effect of Different Computer-aided Design/Computer-aided Manufacturing (CAD/CAM) Materials and Thicknesses on the Fracture Resistance of Occlusal Veneers.

The aim was to evaluate, in vitro, the influence of different computer-aided design/computer-aided manufacturing (CAD/CAM) materials (IPS e.max CAD, Vita Enamic, and Lava Ultimate) and thicknesses (0.6 mm and 1.5 mm) on the fracture resistance of occlusal veneers. Sixty human third molars were prepared to simulate advanced erosion of the occlusal surface, and the teeth were randomly divided into six experimental groups (n=10) according to the material and thickness used to build the veneers. Ten sound teeth formed the control group. The veneers were adhesively luted and submitted to mechanical cyclic loading (1 million cycles at 200-N load). The fracture resistance test was performed in a universal testing machine. The failures were classified as "reparable" and "irreparable." According to two-way analysis of variance and the Tukey test, the interaction (material × thickness) was significant ( p=0.013). The highest fracture resistance was obtained for IPS e.max CAD at a 1.5-mm thickness (4995 N) and was significantly higher compared to the other experimental groups ( p<0.05). The lowest fracture resistance was obtained for Vita Enamic at 0.6 mm (2973 N), although this resistance was not significantly different from those for IPS e.max CAD at 0.6 mm (3067 N), Lava Ultimate at 0.6 mm (3384 N), Vita Enamic at 1.5 mm (3540 N), and Lava Ultimate at 1.5 mm (3584 N) ( p>0.05). The experimental groups did not differ significantly from the sound teeth (3991 N) ( p>0.05). The failures were predominantly repairable. The occlusal veneers of IPS e.max CAD, Vita Enamic, and Lava Ultimate, with thicknesses of 0.6 mm and 1.5 mm, obtained fracture resistances similar to those associated with sound teeth.

Alemtuzumab as graft-versus-host disease (GVHD) prophylaxis strategy in a developing country: lower rate of acute GVHD, increased risk of cytomegalovirus reactivation.

Acute graft-versus-host disease (aGVHD) and cytomegalovirus reactivation are important complications after allogeneic stem cell transplantation (alloHSCT). Here, we evaluated the impact of treatment with alemtuzumab on the occurrence of aGVHD, cytomegalovirus reactivation and survival after alloHSCT. This was a prospective cohort study conducted at the allo-HSCT unit of Hospital das Clínicas, Universidade Federal de Minas Gerais, Brazil, from January 2009 to December 2011. Fifty-seven patients who underwent alloHSCT were included. Forty-five (79%) patients had a malignant disease. Alemtuzumab was administered before the conditioning regimen at a dose of 1 mg/kg in children and 30 mg/day for 2 days in adults or children weighing more than 40 kg (a total dose of 60 mg) with a non-malignant disease or patients with a malignant disease and high-risk for GVHD mortality. Alemtuzumab was used in 23 (40%) patients, of whom 17 received a reduced-intensity conditioning. Eleven patients presented aGVHD (grade 2-4) and only 1 of them received alemtuzumab. Cumulative incidence of aGVHD (grade 2-4) at day 100 after transplantation (D+100) was 4 for patients receiving alemtuzumab and 29% for patients not receiving alemtuzumab. Cumulative incidence of cytomegalovirus reactivation for patients receiving or not alemtuzumab was 62 and 38%, respectively. Sixteen patients died in the first 100 days after alloHSCT, most of them due to bacterial sepsis. Only 2 patients died of aGVHD until D+100. Overall survival was 50% without any impact of alemtuzumab. Alemtuzumab effectively controlled aGVHD but increased the risk of cytomegalovirus reactivation without improving survival.

Influence of glutathione S-transferase gene polymorphisms on busulfan pharmacokinetics and outcome of hematopoietic stem-cell transplantation in thalassemia pediatric patients.

Hematopoietic stem-cell transplantation (HSCT) is currently the only curative therapeutic option for the treatment of thalassemia. In spite of the high cure rate, HSCT can lead to life-threatening adverse events in some patients. Busulfan (Bu) is a key component of the conditioning regimen prior to HSCT. Inter-individual differences in Bu pharmacokinetics (PK) are hypothesized to influence Bu efficacy and toxicity. Since Bu is mainly metabolized by glutathione S-transferase (GST), we investigated the relationship of GSTA1 and GSTM1 genotypes with first-dose PK and HSCT outcomes in 44 children with thalassemia intermedia and thalassemia major. All children received a myeloablative conditioning regimen with IV Bu. Association analysis revealed a relationship between GSTA169C>T (or haplotype *A/*B) and first Bu dose PK that was dependent on sex and Pesaro risk classification (PRC). Among female patients and patients with PRC I-II, homozygous individuals for the GSTA1T-69 allele defining haplotype *B, had higher Bu exposure and lower clearance (P⩽0.01). Association with HSCT outcomes showed that patients with the GSTM1 null genotypes had higher occurrence of regimen-related toxicity (P=0.01). These results suggest that GST genotypes could be useful to tailor the first Bu dose accordingly to improve HSCT outcome.

Importance of SH groups in catalysis by bovine brain acid phosphatase.

The rate of inactivation of acid phosphatase (EC 3.1.3.2) from bovine brain by dithiobis-(2-nitrobenzoic acid) (Nbs2) is identical to the rate of titration of one of the two SH groups of this enzyme. The rate of inactivation of the enzyme by Nbs2 is pH dependent and, at 300 mM NaCl, can be described by the reaction of a single SH group of pK 8.4. At low ionic strength the pK determined from the k inactivation vs. pH profile is 7.7 and the results deviate markedly from the predicted values at pH values less than or equal to 6. The decrease of V upon addition of salts is paralleled by the decrease of inactivation rate by Nbs2. The relevance of SH groups in catalysis by bovine brain acid phosphatase is discussed in terms of these data.

Activation of low molecular weight acid phosphatase from bovine brain by purines and glycerol.

Low molecular weight acid phosphatase (orthophosphoric monoester phosphophydrolase (acid optimum), EC 3.1.3.2) from bovine brain is activated up to 4-fold by guanosine, guanine, adenine, adenosine, and 6-ethylmercapto-purine. Several pyrimidines and other purines were tested and did not show any activation effect. The rate enhancement induced by purines is uncompetitive and not caused by transphosphorylation to the activator. Using transphosphorylation to glycerol as a probe, it is proposed that the activator binds to one of the phosphorylated intermediates in the reaction pathway. These findings are discussed in terms of the catalytic mechanism of low molecular weight acid phosphatase.

Steady state kinetics and effect of SH inhibitors on acid phosphatase from bovine brain.

1. Product inhibition studies and transphosphorylation to methanol using two different substrates indicate that acid phosphatase from bovine brain forms a phosphoryl enzyme and that the phosphorylation step can not be rate limiting. 2. Acid phosphatase from bovine brain is inhibited by 5,5'-dithiobis-(2-nitrobenzoic acid); this inhibition can be counteracted by inorganic phosphate. Incubation of the enzyme with p-nitrophenyl phosphate in the presence of p-chloromercuribenzoate leads, initially, to a higher degree of inhibition than that found with the same concentration of inhibitor in the absence of substrate. Both the titration by 5,5'-dithiobis-(2-nitrobenzoic acid) and inhibition by p-chloromercuribenzoate are consistant with the presence of 2 SH groups per mol of enzyme.

Granulocyte colony-stimulating factor for poor graft function after allogeneic stem cell transplantation: 3 days of G-CSF identifies long-term responders.

Poor graft function (PGF) is a frequent cause of morbidity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). To study the value of granulocyte colony-stimulating factor (G-CSF) in PGF, we retrospectively analyzed 81 episodes of PGF in 66 patients transplanted from 01/94 to 01/99 from an HLA-identical sibling (n = 45) or an unrelated (n = 21) donor. Median age was 29 years, 55 patients had malignancies. A total of 11 patients received a CD34+ selected graft. Viral infections (25%), myelotoxic drug (33%), fungal/bacterial infections (14%), and GVHD (31%) were present before PGF diagnosis. Median time from allo-HSCT to PGF was 75 (25-474) days. All patients were treated with G-CSF. In 77/81 episodes, there was a response that was sustained in 57. A total of 27 patients presented an increase of white cell count (WBC) >0.1 x 10(9)/l after 3 days of G-CSF. The 5-year survival was 37% and was significantly better in patients with increased WBC > 0.1 x 10(9)/l after 3 days of G-CSF (65 vs 18%, P < 0.0001). In multivariate analysis, increased WBC > 0.1 x 10(9)/l after 3 days of G-CSF (P = 0.002) was associated with better survival, while BuCy-based conditioning (P = 0.02) and GVHD (P = 0.005) were associated with higher risk of death. In conclusion, hematological response after 3 days with G-CSF predicted a better survival for patients with PGF after allo-SCT.

Differential vulnerability of the rat retina, suprachiasmatic nucleus and intergeniculate leaflet to malnutrition induced during brain development.

We investigated in young rats the effects of malnutrition on the main structures of the circadian timing system: retina, hypothalamic suprachiasmatic nuclei (SCN), thalamic intergeniculate leaflet, retinohypothalamic- and geniculohypothalamic tracts. Control rats were born from mothers fed a commercial diet since gestation, and malnourished rats from mothers fed a multideficient diet since gestation (GLA group) or lactation (LA group). After weaning, pups received the same diet as their mothers, and were analysed at postnatal days 27, 30-33 and 60-63. Brain sections were processed to visualise in the SCN neuropeptide Y immunoreactivity and terminal labeling after intraocular tracer injections. Nissl staining was used to assess cytoarchitectonic boundaries of the SCN and cell features in retinal whole mounts. Cell counts, morphometric and densitometric analysis were performed. Compared with controls, the total retinal surface was reduced and the topographical distribution of retinal ganglion cells was altered in malnourished rats, with changes in their density. Alterations were also detected in the SCN dimensions in the GLA and LA groups at one and two postnatal months, as well as in the SCN portion occupied by the retinal input in the GLA group at days 30-33, but not in the NPY-containing geniculohypothalamic tract. The present data point to subtle changes, with a low and differential vulnerability to early malnutrition, of structures involved in circadian timing regulation. Furthermore, the present findings suggest that the altered circadian rhythmicity previously documented in malnourished rats cannot be ascribed to impaired development of the retino- and geniculohypothalamic projections to the SCN.

Influence of bone marrow graft lymphocyte subsets on outcome after HLA-identical sibling transplants.

OBJECTIVE: The aim of this study was to analyze bone marrow lymphocyte subsets and CD34 cell dose and their influence on the outcomes of bone marrow transplantation. MATERIALS AND METHODS: Forty-eight patients (median age 30 years, range 5-54) receiving HLA-identical sibling bone marrow transplantation for hematologic malignancies were analyzed. RESULTS: Median number (range) of nucleated cells and CD34+ cells infused were 2.4 (0.4-6.0) x 10(8)/kg and 3.5 (0.5-13.0) x 10(6)/kg, respectively. Probability of neutrophil recovery was 97%. In a multivariate analysis, time to neutrophil recovery was shortened when a higher number of CD3/CD8 cells was infused (> or =1.0 x 10(7)/kg) (hazard ratio [HR] = 2.13, p = 0.018); when the patient was female or had negative cytomegalovirus serology (HR = 2.03, p = 0.03; HR = 0.41, p = 0.009; respectively). The incidence of grade II to IV acute graft-vs-host disease (GVHD) was 47%. Infusion of >1 x 10(7) CD4 infused/kg increased the risk of acute GVHD (HR = 2.86, p = 0.03). Nineteen of 40 patients at risk experienced chronic GVHD, the risk of which was increased by diagnosis of chronic leukemia (p = 0.03), <2.0 x 10(8) nucleated cells infused/kg (p = 0.05), and a low number of all lymphocyte subsets, except CD19. Estimated 3-year survival rate was 54%. Risk of death was increased in patients receiving <3.5 x 10(6)CD34 infused/kg (HR = 0.37, p = 0.02). Only six patients relapsed. CONCLUSIONS: A high cell dose of CD3/CD8 is associated with faster neutrophil recovery, whereas a high cell dose of CD4+ cells increases the incidence of acute GVHD. A high number of nucleated cells and CD34+ cells infused was associated with decreased risk of chronic GVHD and improved survival, respectively.