Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

Iron overload is one of the secondary osteoporosis etiologies. Cellular and molecular mechanisms involved in iron-related osteoporosis are not fully understood. AIM: The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. MATERIAL AND METHODS: We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. RESULTS: Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 µM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. CONCLUSION: Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

Synergy of a STING agonist and an IL-2 superkine in cancer immunotherapy against MHC I-deficient and MHC I+ tumors.

Cyclic dinucleotides (CDN) and Toll-like receptor (TLR) ligands mobilize antitumor responses by natural killer (NK) cells and T cells, potentially serving as complementary therapies to immune checkpoint therapy. In the clinic thus far, however, CDN therapy targeting stimulator of interferon genes (STING) protein has yielded mixed results, perhaps because it initiates responses potently but does not provide signals to sustain activation and proliferation of activated cytotoxic lymphocytes. To improve efficacy, we combined CDN with a half life-extended interleukin-2 (IL-2) superkine, H9-MSA (mouse serum albumin). CDN/H9-MSA therapy induced dramatic long-term remissions of the most difficult to treat major histocompatibility complex class I (MHC I)­deficient and MHC I+ tumor transplant models. H9-MSA combined with CpG oligonucleotide also induced potent responses. Mechanistically, tumor elimination required CD8 T cells and not NK cells in the case of MHC I+ tumors and NK cells but not CD8 T cells in the case of MHC-deficient tumors. Furthermore, combination therapy resulted in more prolonged and more intense NK cell activation, cytotoxicity, and expression of cytotoxic effector molecules in comparison with monotherapy. Remarkably, in a primary autochthonous sarcoma model that is refractory to PD-1 checkpoint therapy, the combination of CDN/H9-MSA with checkpoint therapy yielded long-term remissions in the majority of the animals, mediated by T cells and NK cells. This combination therapy has the potential to activate responses in tumors resistant to current therapies and prevent MHC I loss accompanying acquired resistance of tumors to checkpoint therapy.

The effect of a natural polyphenol supplement on iron absorption in adults with hereditary hemochromatosis.

OBJECTIVES: We developed a natural polyphenol supplement that strongly chelates iron in vitro and assessed its effect on non-heme iron absorption in patients with hereditary hemochromatosis (HH). METHODS: We performed in vitro iron digestion experiments to determine iron precipitation by 12 polyphenol-rich dietary sources, and formulated a polyphenol supplement (PPS) containing black tea powder, cocoa powder and grape juice extract. In a multi-center, single-blind, placebo-controlled cross-over study, we assessed the effect of the PPS on iron absorption from an extrinsically labelled test meal and test drink in patients (n = 14) with HH homozygous for the p.C282Y variant in the HFE gene. We measured fractional iron absorption (FIA) as stable iron isotope incorporation into erythrocytes. RESULTS: Black tea powder, cocoa powder and grape juice extract most effectively precipitated iron in vitro. A PPS mixture of these three extracts precipitated ~ 80% of iron when 2 g was added to a 500 g iron solution containing 20 µg Fe/g. In the iron absorption study, the PPS reduced FIA by ~ 40%: FIA from the meal consumed with the PPS was lower (3.01% (1.60, 5.64)) than with placebo (5.21% (3.92, 6.92)) (p = 0.026)), and FIA from the test drink with the PPS was lower (10.3% (7.29 14.6)) than with placebo (16.9% (12.8 22.2)) (p = 0.002). CONCLUSION: Our results indicate that when taken with meals, this natural PPS can decrease dietary iron absorption, and might thereby reduce body iron accumulation and the frequency of phlebotomy in patients with HH. TRIAL REGISTRY: clinicaltrials.gov (registration date: 9.6.2019, NCT03990181).

Cordycepin Sensitizes Cholangiocarcinoma Cells to Be Killed by Natural Killer-92 (NK-92) Cells.

Immunotherapy harnessing immune functions is a promising strategy for cancer treatment. Tumor sensitization is one approach to enhance tumor cell susceptibility to immune cell cytotoxicity that can be used in combination with immunotherapy to achieve therapeutic efficiency. Cordycepin, a bioactive compound that can be extracted from some Cordyceps spp. has been reported to effectively inhibit tumor growth, however, the mechanism of its tumor sensitization activity that enhances immune cell cytotoxicity is unknown. In the present study, we investigated the potency of cordycepin to sensitize a lethal cancer, cholangiocarcinoma (CCA), to natural killer (NK) cells. Treatment with cordycepin prior to and during co-culturing with NK-92 cells significantly increased cell death of KKU-213A as compared to solitary cordycepin or NK treatment. Moreover, sensitization activity was also observed in the combination of NK-92 cells and Cordyceps militaris extract that contained cordycepin as a major component. The cordycepin treatment remarkably caused an increase in TRAIL receptor (DR4 and DR5) expression in KKU-213A, suggesting the possible involvement of TRAIL signaling in KKU-213A sensitization to NK-92 cells. In conclusion, this is the first report on the sensitization activity of cordycepin on CCA cells to NK cytotoxicity, which supports that cordycepin can be further developed as an alternate immunomodulating agent.

Combination of cassette-dosing and microsampling for reduced animal usage for antibody pharmacokinetics in cynomolgus monkeys, wild-type mice, and human FcRn transgenic mice.

PURPOSE: The aim of this study was to develop a useful antibody PK evaluation tool using a combination of cassette-dosing and microsampling in mice and monkeys in order to reduce the number of animals used. METHODS: Cetuximab, denosumab, infliximab, and a mixture of the three antibodies, i.e., cassette-dosing, were administered intravenously to cynomolgus monkeys, C57BL/6J mice, and homozygous human neonatal Fc-receptor transgenic (Tg32) mice. Mouse blood was collected from one animal continuously via the jugular vein at nine points. RESULTS: In cynomolgus monkeys, infliximab showed faster elimination in the cassette-dosing group than in the single-dose group. Anti-drug antibody production was observed, but the PK parameters of the clearance and distribution volume were similar in both groups. In C57BL/6J and Tg32 mice, each of the plasma concentrations-time profiles after cassette-dosing were similar to those after single dosing. PK evaluation using a combination of cassette-dosing and microsampling in mice may reduce the number of mice used by approximately 90% compared with the conventional method. CONCLUSIONS: The combination of antibody cassette-dosing and microsampling is a promising PK evaluation method as a high-throughput and reliable with reduced numbers of mice and cynomolgus monkeys.

FcRn is not the receptor mediating the transfer of serum IgG to colostrum in pigs.

In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR-Cas9-based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.

Prediction of Human Pharmacokinetics Profile of Monoclonal Antibody Using hFcRn Transgenic Mouse Model.

Human pharmacokinetics (PK) profiles of monoclonal antibodies (mAbs) are usually predicted using non-human primates (NHP), but this comes with drawbacks in terms of cost and throughput. Therefore, we established a human PK profile prediction method using human neonatal Fc receptor (hFcRn) transgenic mice (TgM). We administered launched 13 mAbs to hFcRn TgM and measured the concentration in plasma using electro-chemiluminescence immunoassay. This was then used to calculate PK parameters and predict human PK profiles. The mAbs showed a bi-phased elimination pattern, and clearance (CL) (mL/d/kg) and distribution volume at steady state (Vdss) (mL/kg) ranges were 11.0 to 131 and 110 to 285, respectively. There was a correlation in half-life at elimination phase (t1/2ß) between hFcRn TgM and humans for 10 mAbs showing CL of more than 80% in the elimination phase (R2 = 0.714). Human t1/2ß was predicted using hFcRn TgM t1/2ß; 9 out of 10 mAbs were within 2-fold the actual values, and all mAbs were within 3-fold. Regarding the predicted CL values, 7 out of 10 mAbs were within 2-fold the human values and all mAbs were within 3-fold. Furthermore, even on day 7 the predicted CL values of 8 out of 10 mAbs were within 2-fold the observed value, with all mAbs within 3-fold. These results suggest human PK profiles can be predicted using hFcRn TgM data. These methods can accelerate the development of antibody drugs while also reducing cost and improving throughput.

Use of Cryopreserved Hepatocytes as Part of an Integrated Strategy to Characterize In Vivo Clearance for Peptide-Antibody Conjugate Inhibitors of Nav1.7 in Preclinical Species.

The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = -0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies. SIGNIFICANCE STATEMENT: Although multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.

Coffee consumption, genetic susceptibility and risk of latent autoimmune diabetes in adults: A population-based case-control study.

AIM: Coffee consumption is inversely related to risk of type 2 diabetes (T2D). In contrast, an increased risk of latent autoimmune diabetes in adults (LADA) has been reported in heavy coffee consumers, primarily in a subgroup with stronger autoimmune characteristics. Our study aimed to investigate whether coffee consumption interacts with HLA genotypes in relation to risk of LADA. METHODS: This population-based study comprised incident cases of LADA (n=484) and T2D (n=1609), and also 885 healthy controls. Information on coffee consumption was collected by food frequency questionnaire. Odds ratios (ORs) with 95% CIs of diabetes were calculated and adjusted for age, gender, BMI, education level, smoking and alcohol intake. Potential interactions between coffee consumption and high-risk HLA genotypes were calculated by attributable proportion (AP) due to interaction. RESULTS: Coffee intake was positively associated with LADA in carriers of high-risk HLA genotypes (OR: 1.14 per cup/day, 95% CI: 1.02-1.28), whereas no association was observed in non-carriers (OR: 1.04, 95% CI: 0.93-1.17). Subjects with both heavy coffee consumption (≥4 cups/day) and high-risk HLA genotypes had an OR of 5.74 (95% CI: 3.34-9.88) with an estimated AP of 0.36 (95% CI: 0.01-0.71; P=0.04370). CONCLUSION: Our findings suggest that coffee consumption interacts with HLA to promote LADA.

Design and preclinical characterization of ALXN1210: A novel anti-C5 antibody with extended duration of action.

Eculizumab, a monoclonal antibody (mAb) directed against complement protein C5, is considered to be the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. This study describes the generation and preclinical attributes of ALXN1210, a new long-acting anti-C5 mAb, obtained through select modifications to eculizumab to both largely abolish target-mediated drug disposition (TMDD) and increase recycling efficiency via the neonatal Fc receptor (FcRn). To attenuate the effect of TMDD on plasma terminal half-life (t1/2), histidine substitutions were engineered into the complementarity-determining regions of eculizumab to enhance the dissociation rate of the mAb:C5 complex in the acidic early endosome relative to the slightly basic pH of blood. Antibody variants with optimal pH-dependent binding to C5 exhibited little to no TMDD in mice in the presence of human C5. To further enhance the efficiency of FcRn-mediated recycling of the antibody, two additional substitutions were introduced to increase affinity for human FcRn. These substitutions yielded an additional doubling of the t½ of surrogate anti-mouse C5 antibodies with reduced TMDD in transgenic mice expressing the human FcRn. In conclusion, ALXN1210 is a promising new therapeutic candidate currently in clinical development for treatment of patients with PNH and atypical hemolytic uremic syndrome.

Identification of MHC Haplotypes Associated with Drug-induced Hypersensitivity Reactions in Cynomolgus Monkeys.

Drug-induced hypersensitivity reactions can significantly impact drug development and use. Studies to understand risk factors for drug-induced hypersensitivity reactions have identified genetic association with specific human leukocyte antigen (HLA) alleles. Interestingly, drug-induced hypersensitivity reactions can occur in nonhuman primates; however, association between drug-induced hypersensitivity reactions and major histocompatibility complex (MHC) alleles has not been described. In this study, tissue samples were collected from 62 cynomolgus monkeys from preclinical studies in which 9 animals had evidence of drug-induced hypersensitivity reactions. Microsatellite analysis was used to determine MHC haplotypes for each animal. A total of 7 haplotypes and recombinant MHC haplotypes were observed, with distribution frequency comparable to known MHC I allele frequency in cynomolgus monkeys. Genetic association analysis identified alleles from the M3 haplotype of the MHC I B region (B*011:01, B*075:01, B*079:01, B*070:02, B*098:05, and B*165:01) to be significantly associated (χ2 test for trend, p < 0.05) with occurrence of drug-induced hypersensitivity reactions. Sequence similarity from alignment of alleles in the M3 haplotype B region and HLA alleles associated with drug-induced hypersensitivity reactions in humans was 86% to 93%. These data demonstrate that MHC alleles in cynomolgus monkeys are associated with drug-induced hypersensitivity reactions, similar to HLA alleles in humans.

Genome-wide gene-based analysis suggests an association between Neuroligin 1 (NLGN1) and post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) develops in only some people following trauma exposure, but the mechanisms differentially explaining risk versus resilience remain largely unknown. PTSD is heritable but candidate gene studies and genome-wide association studies (GWAS) have identified only a modest number of genes that reliably contribute to PTSD. New gene-based methods may help identify additional genes that increase risk for PTSD development or severity. We applied gene-based testing to GWAS data from the Grady Trauma Project (GTP), a primarily African American cohort, and identified two genes (NLGN1 and ZNRD1-AS1) that associate with PTSD after multiple test correction. Although the top SNP from NLGN1 did not replicate, we observed gene-based replication of NLGN1 with PTSD in the Drakenstein Child Health Study (DCHS) cohort from Cape Town. NLGN1 has previously been associated with autism, and it encodes neuroligin 1, a protein involved in synaptogenesis, learning, and memory. Within the GTP dataset, a single nucleotide polymorphism (SNP), rs6779753, underlying the gene-based association, associated with the intermediate phenotypes of higher startle response and greater functional magnetic resonance imaging activation of the amygdala, orbitofrontal cortex, right thalamus and right fusiform gyrus in response to fearful faces. These findings support a contribution of the NLGN1 gene pathway to the neurobiological underpinnings of PTSD.

Human FcRn Transgenic Mice for Pharmacokinetic Evaluation of Therapeutic Antibodies.

Therapeutic monoclonal antibodies are widely recognized to be a most promising means to treat an increasing number of human diseases, including cancers and autoimmunity. To a large extent, the efficacy of monoclonal antibody treatment is because IgG antibodies have greatly extended persistence in vivo. However, conventional rodent models do not mirror human antibody pharmacokinetics. The key molecule responsible for the extended persistence antibodies is the major histocompatibility complex class I family Fc receptor, FcRn. We describe human FcRn transgenic mouse models and how they can be exploited productively for the preclinical pharmacokinetic evaluation of therapeutic antibodies.

Haemochromatosis.

Haemochromatosis is now known to be an iron-storage disease with genetic heterogeneity but with a final common metabolic pathway resulting in inappropriately low production of the hormone hepcidin. This leads to increase in intestinal absorption and deposition of excessive amounts of iron in parenchymal cells which in turn results in eventual tissue damage and organ failure. A clinical enigma has been the variable clinical expression with some patients presenting with hepatic cirrhosis at a young age and others almost asymptomatic for life. Research is unravelling this puzzle by identifying environmental factors-especially alcohol consumption-and associated modifying genes that modulate phenotypic expression. A high index of suspicion is required for early diagnosis but this can lead to presymptomatic therapy and a normal life expectancy. Venesection (phlebotomy) therapy remains the mainstay of therapy, but alternative therapies are the subject of current research.

Hemochromatosis: Niche Construction and the Genetic Domino Effect in the European Neolithic.

Hereditary hemochromatosis is caused by a potentially lethal recessive gene (HFE, C282Y allele) that increases iron absorption and reaches polymorphic levels in northern European populations. Because persons carrying the allele absorb iron more readily than do noncarriers, it has often been suggested that HFE is an adaptation to anemia. We hypothesize positive selection for HFE began during or after the European Neolithic with the adoption of an iron-deficient high-grain and dairying diet and consequent anemia, a finding confirmed in Neolithic and later European skeletons. HFE frequency compared with rate of lactase persistence in Eurasia yields a positive linear correlation coefficient of 0.86. We suggest this is just one of many mutations that became common after the adoption of agriculture.

The effects of okra (Abelmoschus esculentus Linn.) on the cellular events associated with Alzheimer's disease in a stably expressed HFE neuroblastoma SH-SY5Y cell line.

It has been reported that persons carrying the H63D variant in their hemochromatosis (HFE) gene are at increased risk of Alzheimer's disease (AD). We investigated the possibility that okra (Abelmoschus esculentus) and quercetin could mitigate this risk factor by examining its effect on AD-associated cellular events in HFE stably expressing SH-SY5Y cells. Treatment of H63D HFE cells either with okra or quercetin significantly decreased reactive oxygen species (ROS), hydrogen peroxide (H2O2), and protein oxidation compared to untreated cells. The levels of tau phosphorylation at serine-199, serine-202, and serine-396 sites were also significantly decreased when cells were treated with okra. Exposure of the H63D and wild type (WT) cells to iron increased tau phosphorylation, but this response was decreased significantly when cells were treated with okra. The mechanism responsible for these changes appears to be related to decreased glycogen synthase kinase (GSK)-3ß activity, an upstream signaling kinase of tau phosphorylation. We also established that okra treatment dramatically decreases intracellular iron levels in H63D cells compared to untreated cells. Our results provide important in vitro data on the effects of okra on various AD-associated cellular processes in H63D variant HFE cells. These results suggest okra may be beneficial in people expressing the H63D variant to reduce the risk of AD and other neurodegenerative diseases related to oxidative stress. Further in vivo studies would help confirm this.

Analysis of Fcgrt gene polymorphism in indigenous Chinese sheep and its association with colostrum IgG concentration.

The neonatal Fc receptor (FcRn) plays an important role in regulating IgG homeostasis in the body and passive protection to the offspring. Changes in FcRn expression levels caused by genetic polymorphisms of Fcgrt, which encodes FcRn, may lead to inter-individual differences in colostrum IgG levels in sheep. In this study, we sequenced the FcRn partial heavy chain from 179 sheep from Xinjiang Province, China, and detected the differences in colostrum IgG levels and Fcgrt genotypes to identify the correlation between the Fcgrt genotype and colostrum IgG levels in 4 sheep breeds. The DNA sequencing of a 680-bp fragment of the Fcgrt gene revealed various patterns depending on the single-strand conformation in the Suffolk breed. Sequencing analysis revealed a total of 3 patterns, AA, BB, AB, in this fragment, among which the absence of AB and BB genotype acted as a marker for breed identification and characterization, while the AA genotype was shared by Suffolk and 3 other breeds. The only allele found in all 4 breeds was allele A, indicating that natural selection may be favoring the AB and BB genotypes in general and B allele in particular, as the colostrum IgG concentration was relatively higher in the Suffolk breed compared to the other 3 breeds.

Deferasirox in patients with iron overload secondary to hereditary hemochromatosis: results of a 1-yr Phase 2 study.

This open-label, prospective, phase 2 study evaluated the safety and efficacy of deferasirox (10 ± 5 mg/kg/d) in patients with hereditary hemochromatosis (HH) and iron overload refractory to or intolerant of phlebotomy. Ten patients were enrolled and all completed the 12-month treatment period. There were significant decreases from baseline to end of study (i.e., 12 months) in median serum ferritin (P < 0.001), mean transferrin saturation (P < 0.05), median liver iron concentration (P < 0.001), and mean alanine aminotransferase (P < 0.05). The median time to achieve serum ferritin reduction ≥50% compared to baseline was 7.53 months. The most common adverse events were mild, transient diarrhea (n = 5) and nausea (n = 2). No patient experienced an increase in serum creatinine that exceeded the upper limit of normal. These data confirm that deferasirox was well tolerated and effective in reducing iron burden in patients with hereditary hemochromatosis and could be a safe alternative to phlebotomy in selected patients.

HLA class I expression and its alteration by preoperative hyperthermo-chemoradiotherapy in patients with rectal cancer.

OBJECTIVE: Enhancing immunologic responses, including human leukocyte antigen (HLA) class I expression on tumor cells and recognition and elimination of tumor cells by tumor-specific cytotoxic T lymphocyte (CTL), is considered a novel concept of radiotherapy. The present study examined patients who underwent preoperative hyperthermo-chemoradiotherapy (HCRT) for locally advanced rectal cancer to assess the correlation between HLA class I expression and clinical outcome. MATERIALS AND METHODS: Seventy-eight patients with locally advanced rectal adenocarcinoma who received preoperative HCRT were enrolled. The median age of the patients was 64 years (range, 33-85 years) and 4, 18, and 56 patients had clinical stage I, II and III disease, respectively. Formalin-fixed and paraffin-embedded tissues excised before and after HCRT were subjected to immunohistochemical analysis with an anti-HLA class I-A, B, C antibody. HLA class I expression was graded according to tumor cell positivity. RESULTS: In pre-HCRT, the number of specimens categorized as Grade 0 and 1 were 19 (24%) and 58 (74%), respectively. Only 1 patient (1%) showed Grade 2 expression. However, 6 (8%), 27 (35%), 7 (9%), and 12 (15%) post-HCRT specimens were graded as Grade 0, 1, 2, and 3, respectively. There was a significant increase in HLA class I expression in post-HCRT specimens (p<0.01). However, neither pre- nor post-HCRT HLA class I expression affected overall survival and distant metastasis-free survival in clinical stage III patients. Univariate analysis revealed that Post-HCRT HLA class I expression showed a significant negative relationship with LC (p<0.05). Nevertheless, multivariate analysis showed that there was no correlation between HLA class I expression and clinical outcome. CONCLUSION: HCRT increased HLA class I expression in rectal cancer patients. However, multivariate analysis failed to show any correlation between the level of HLA class I expression and prognosis.

Adapting iron dose supplementation in pregnancy for greater effectiveness on mother and child health: protocol of the ECLIPSES randomized clinical trial.

BACKGROUND: Currently, there is no consensus regarding iron supplementation dose that is most beneficial for maternal and offspring health during gestation. Recommended iron supplementation dose does not preempt anemia in around 20% of the pregnancies, nor the risk of hemoconcentration in 15%. This deficit, or excess, of iron prejudices the mother-child wellbeing. Therefore the aims of the study are to determine the highest level of effectiveness of iron supplementation adapted to hemoglobin (Hb) levels in early pregnancy, which would be optimum for mother-child health. DESIGN: Randomized Clinical Trial (RCT) triple-blindedSetting: 10 Primary Care Centers from Catalunya (Spain)Study subjects: 878 non-anemic pregnant women at early gestation stage, and their subsequent newborns METHODS: The study is structured as a RCT with 2 strata, depending on the Hb levels before week 12 of gestation. Stratum #1: If Hb from 110 to 130 g/L, randomly assigned at week 12 to receive iron supplement of 40 or 80 mg/d. Stratum #2: If Hb >130 g/L, randomly assigned at week 12 to receive iron supplement of 40 or 20 mg/d. MEASUREMENTS: In the mother: socio-economic data, clinical history, food item frequency, lifestyle and emotional state, and adherence to iron supplement prescription. Biochemical measurements include: Hb, serum ferritin, C reactive protein, cortisol, and alterations in the HFE gene (C282Y, H63D). In children: ultrasound fetal biometry, anthropometric measurements, and temperament development.Statistical analyses, using the SPSS program for Windows, will include bivariate and multivariate analyses adjusted for variables associated with the relationship under study. DISCUSSION: Should conclusive outcomes be reached, the study would indicate the optimal iron supplementation dose required to promote maternal and infant health. These results would contribute towards developing guidelines for good clinical practice.