Prevalence of thrombotic risk factors among β-thalassemia patients from Western Iran

Background There is evidence for increased risk of thrombosis in patients with β-thalassemia intermedia and β-thalassemia major. The present study investigated the prevalence of thromboembolic risk factors of prothrombin G20210A, factor V Leiden G1691A and methylentetrahydrofolate reductase (MTHFR) C677T, as well as the hematological and clinical profiles in β-thalassemia major and intermedia patients from Western Iran. Methods Patients consisted of 158 β-thalassemia patients, 151 β-thalassemia major and 7 β-thalassemia intermedia patients, including 82 males and 76 females aged 13.6 ± 6.3 years. The control group were 180 healthy blood donors and school students, consisting of 103 males and 77 females aged 16.8 ± 2.1. Genotyping was done by PCR-RFLP using Mnl I, Hind III and Hinf I for factor V Leiden and prothrombin G20210A and MTHFR, respectively. Results The prevalence of prothrombin G20210A variant in patients and healthy individuals were 1.3 and 3.3%, respectively. Factor V Leiden G1691A was insignificantly higher in β-thalassemia patients (prevalence 5.7% and allele frequency 3.2%) compared to healthy individuals (2.8%). This mutation was found in eight β-thalassemia major (5.3%) and one β-thalassemia intermedia (14.3%) patients. The prevalence of MTHFR C677T polymorphism was slightly higher in patients (50%) compared to healthy individuals (48.3%). Around 71% of β-thalassemia intermedia and 38.4% of β-thalassemia major patients had undergone splenectomy. In β-thalassemia major patients, 5.3% had insulin dependent diabetes mellitus (IDDM) and 6.6% had HCV antibodies. All patients with IDDM were splenectomized and in one of them the prothrombin G20210A variant was found. Two patients, a 7-year-old boy with β-thalassemia intermedia receiving regularly blood transfusion and a β-thalassemia major patient (a 22-year-old splenectomized female), were found to be homozygous for MTHFR 677TT and heterozygous for factor V Leiden G1691A. Double heterozygosity for factor V Leiden G1691A and MTHFR C677T and also homozygous factor V Leiden 1691AA were found in two β-thalassemia major patients. No thromboembolic event has been recorded in the files of patients. Conclusions The results of present study establish the prevalence of biological risk factors of thrombosis in β-thalassemia patients from Western Iran. It seems that thrombophilic mutations may not be associated with thrombotic events in thalassemic patients, which needs to be confirmed by the study of larger sample sizes.     

Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with <Superscript>111</Superscript>In using N-terminal DOTA,NOTA and NODAGA chelators in mice bearing prostate cancer xenografts

Purpose  

In disseminated prostate cancer, expression of human epidermal growth factor receptor type 2 (HER2) is one of the pathways to androgen independence. Radionuclide molecular imaging of HER2 expression in disseminated prostate cancer might identify patients for HER2-targeted therapy. Affibody molecules are small (7 kDa) targeting proteins with high potential as tracers for radionuclide imaging. The goal of this study was to develop an optimal Affibody-based tracer for visualization of HER2 expression in prostate cancer.     

Burr-hole drainage for the treatment of acute epidural hematoma in coagulopathic patients: a report of eight cases

Craniotomy has been accepted as the treatment of choice for the management of acute epidural hematomas (AEDH). However, in practice, it seems possible to evacuate AEDH via a single burr hole instead of the traditional craniotomy in certain circumstances. Among 160 patients with AEDH meeting criteria for evacuation admitted to the emergency and accident division of our center between 2006 and 2009, we found 8 cases of hematoma appearing isodense to brain parenchyma on computed tomography (CT), who had concomitant coagulopathy. These patients were managed by burr-hole drainage for treatment of the liquefied AEDH. A closed drainage system was then kept in the epidural space for 3 days. In all 8 patients, AEDH was evacuated successfully via burr-hole placement over the site of hematoma. The level of consciousness and other symptoms improved within the first day, and no patient required an additional routine craniotomy. For patients with slowly-developing AEDH in the context of impaired coagulation, burr-hole evacuation and drainage might be a less invasive method of treatment compared to conventional craniotomy.     

Recombinant Nonstructural 3 Protein,rNS3, of Hepatitis C Virus Along With Recombinant GP96 Induce IL-12, TNFα and α5integrin Expression in Antigen Presenting Cells

Background

Hepatitis C virus (HCV) infection is the main cause of chronic liver disease and to date there has been no vaccine development to prevent this infection. Among non-structural HCV proteins, NS3 protein is an excellent goal for a therapeutic vaccine, due to its large size and less variation in conserved regions. The immunogenic properties of heat shock proteins (HSPs) for instance GP96 have prompted investigations into their function as strong adjuvant to improve innate and adaptive immunity.

Objectives

The aim of this study was to examine additive effects of recombinant GP96 (rGP96) fragments accompanied by rNS3 on expression levels of α5integrin and pro-inflammatory cytokines, IL-12 and TNFα, in Antigen Presenting Cells (APCs).

Materials and Methods

Recombinant viral proteins (rNS3 and rRGD-NS3), N-terminal and C-terminal fragments of GP96 were produced and purified from E. coli in order to treat the cells; mouse spleen Dendritic Cells (DCs) and THP-1 macrophages.

Results

Our results showed that rNT-GP96 alone significantly increases the expression level of IL-12, TNFα and α5integrin in THP-1 macrophages and DCs, while IL-12 and TNFα expression levels were unaffected by either rNS3 or rRGD-NS3. Interestingly, the co-addition of these recombinant proteins with rNT-GP96 increased IL-12, TNFα and α5integrin expression. Pearson Correlation showed a direct association between α5integrin with IL-12 and TNF-α expression.

Conclusions

we have highlighted the role of rNS3 plus rNT-GP96 mediated by α5integrin in producing IL-12 and TNFα. It can be suggested that rNT-GP96 could enhance immunity characteristic of rNS3 protein via production of pro-inflammatory cytokines.     

The β-GLobin Gene Haplotypes Associated With Hb D-Los Angeles [β121(GH4)Glu→Gln] in Western Iran

Hb D-Los Angeles is characterized by the substitution of glutamine for glutamic acid at position 121 of the β-globin chain. The present investigation is the first study on the β-globin gene haplotypes associated with β-D-Los Angeles in Western Iran. Thirty two individuals from 11 unrelated families from Western Iran were studied. The Hb D-Los Angeles status of all cases was confirmed using polymerase chain reaction (PCR) followed by digestion with EcoRI. The haplotype of the β‐globin gene cluster was determined by a PCR-RFLP (restriction fragment length polymorphism) procedure. The haplotype background of the β^A chromosomes was also determined in 35 normal subjects from the same geographic region. The β-globin gene haplotype analysis demonstrated that all β-D-Los Angeles genes (23 genes) were in linkage disequilibrium with haplotype I [+????++]. Among the 70 β^A chromosomes, 30 chromosomes (42.9%) were associated with haplotype I. The present study indicates the unicentric origin of the β-D-Los Angeles gene in Western Iran. It seems that this mutation may have occurred at the same chromosomal background common in the local population.     

Dendritic cell paucity in mismatch repair–proficient colorectal cancer liver metastases limits immune checkpoint blockade efficacy

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair–proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC.

Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment in recent years. Anti–PD1 (anti–programmed cell-death protein 1) and anti–CTLA-4 (anti–cytotoxic T lymphocyte-associated protein 4) are two main types of ICB therapies that can be particularly effective (1). Metastatic melanoma, which was previously an incurable disease, now has cure rates of more than 50% when patients are treated with a combination of anti–PD1 and anti–CTLA-4 (2). However, ICB therapy is only effective in less than 15% of patients who receive the therapy (3), and efforts are ongoing to uncover the underlying mechanisms of intrinsic and acquired resistance.Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States (4) and in the world (5). Metastatic spread, especially to the liver, is a major cause of mortality in patients with CRC (6). The efficacy of ICB therapy in metastatic CRCs has been limited to patients with mismatch repair–deficient (dMMR) or microsatellite instability-high (MSI-H) tumors, where a 55% objective response rate has been achieved (7). However, dMMR or MSI-H metastatic CRCs represent only about 5% of total metastatic CRC cases. The remaining 95% are mismatch repair–proficient (pMMR) or microsatellite stable (MSS) tumors (8), which are typically unresponsive to ICB therapy (8). Therefore, there is an urgent need to better understand the resistance mechanisms in pMMR and MSS metastatic CRCs, and improve the efficacy of treatments against this disease.Preclinical mouse models of cancer are effective tools for studying and improving cancer therapy. MC38 and CT26 are syngeneic mouse CRC cell lines commonly used in preclinical immunocompetent mouse models of cancer. In most preclinical studies, these cells are injected under the skin into the hind flank of mice, where they grow as subcutaneous tumors. When treated with ICB therapies such as anti–PD1 and/or anti–CTLA-4, these tumors have been shown to respond well (9, 10). However, MC38 and CT26 lack coding somatic mutations in the DNA mismatch repair genes and should be considered as pMMR CRC cell lines (11, 12). Hence, experimental preclinical models using subcutaneously implanted pMMR CRC cell lines fail to recapitulate the disease resistance to ICB therapy that is observed in patients.We hypothesized that orthotopic pMMR CRC mouse models, where pMMR CRC cells are implanted in the colon to represent primary colon tumors or in the liver to represent liver metastases, would more accurately recapitulate progression of the human disease and its response to ICB treatment in the clinic. Indeed, we report here strikingly different sensitivities to ICB treatment for pMMR CRC tumors grown orthotopically when compared with their subcutaneous counterparts. We further take advantage of these differences to define local nonmalignant components that determine the sensitivity of pMMR CRCs to treatment.     

Avoidance and escape conditioning adjust adult neurogenesis to conserve a fit hippocampus in adult male rodents

In this study, the connection between cognitive behaviors and the adult rodent hippocampus was investigated. Recording field potentials at performant pathway (PP)–hippocampal dentate gyrus (DG) synapses in transverse slices from the dorsal (d), intermediate (i), and ventral (v) hippocampus showed differences in paired-pulse responses and long-term potentiation in rats. The Barnes maze (BM) and passive avoidance (PA) tests indicated a decrease in escape latency and step-through latency in both rats and mice over training days. A decrease in the use of random or sequential strategy while an increase in the use of direct strategy to search for an escape box occurred in both groups. Evaluation of the levels of neurogenesis markers (Ki67 and BrdU/NeuN) by immunofluorescence assay in the dDG, iDG, and vDG revealed a long-axis disparity in the hippocampal dentate baseline cell proliferation and exposure to the BM and PA task changed the profile of baseline cell proliferation along the DG in both rats and mice. Also, these learning experiences changed the profile of BrdU⁺/NeuN⁺ cells along the DG of rats. Quantitation of hippocampal BDNF protein levels using ELISA exhibited no changes in BDNF levels due to learning experiences in rats. We demonstrate that PP–DG synaptic efficacy and neurogenesis are organized along a gradient. Avoidance and escape conditioning themselves are sufficient to change and calibrate adult neurogenesis along the hippocampal long axis in rodents. Further research will be required to determine the precise mechanisms underlying the role of experience-derived neuroplasticity in cognitive function and decline.     

Effects of Papaver rhoeas extract on the acquisition and expression of morphine-induced behavioral sensitization in mice

In the present study, the effects of a water-alcohol extract of Papaver rhoeas on the acquisition and expression of morphine-induced behavioral sensitization in mice were investigated. The subcutaneous (s.c.) administration of morphine (50 mg/kg) induced locomotor activity in animals, whereas the drug did not show an effect at a dose of 5 mg/kg. On the other hand, intraperitoneal (i.p.) administration of the plant extract (25, 50 and 100 mg/kg) did not show any effect. The locomotor behavioral response was enhanced in mice pretreated with morphine (5 mg/kg, daily x 3 days) alone, indicating that sensitization had developed. Extract (25, 50 and 100 mg/kg, i.p.) administration, 30 min before each of the three daily doses of morphine decreased the development of sensitization. Moreover, intraperitoneal administration of the plant extract (25, 50 and 100 mg/kg) 30 min before the test reduced the expression of morphine-induced behavioral sensitization.The results indicate that administration of the extract of Papaver rhoeas reduced the acquisition and expression of morphine-induced behavioral sensitization in mice.