Therapeutic application of Sertoli cells for treatment of various diseases.

Sertoli cells (SCs) are immune privileged cells found in the testis that function to immunologically protect maturing germ cells from immune destruction. This immune protection is due to the blood-testis-barrier, which prevents infiltration of cytotoxic immune cells and antibodies, and SC production of immunomodulatory factors, that favor a tolerogenic environment. The ability of SCs to create an immune privileged environment has led to the exploration of their potential use in the treatment of various diseases. SCs have been utilized to create a tolerogenic ectopic microenvironment, to protect co-grafted cells, and to deliver therapeutic proteins through gene therapy. To date, numerous studies have reported the potential use of SCs for the treatment of diabetes, neurodegenerative disorders, and restoration of spermatogenesis. Additionally, SCs have been investigated as a delivery vehicle for therapeutic products to treat other diseases like Laron syndrome, muscular dystrophy, and infections. This review will provide an overview of these therapeutic applications.

Integrated single-cell transcriptome analysis of CD34 + enriched leukemic stem cells revealed intra- and inter-patient transcriptional heterogeneity in pediatric acute myeloid leukemia.

To gain insights into the idiosyncrasies of CD34 + enriched leukemic stem cells, we investigated the nature and extent of transcriptional heterogeneity by single-cell sequencing in pediatric AML. Whole transcriptome analysis of 28,029 AML single cells was performed using the nanowell cartridge-based barcoding technology. Integrated transcriptional analysis identified unique leukemic stem cell clusters of each patient and intra-patient heterogeneity was revealed by multiple LSC-enriched clusters differing in their cell cycle processes and BCL2 expression. All LSC-enriched clusters exhibited gene expression profile of dormancy and self-renewal. Upregulation of genes involved in non-coding RNA processing and ribonucleoprotein assembly were observed in LSC-enriched clusters relative to HSC. The genes involved in regulation of apoptotic processes, response to cytokine stimulus, and negative regulation of transcription were upregulated in LSC-enriched clusters as compared to the blasts. Validation of top altered genes in LSC-enriched clusters confirmed upregulation of TCF7L2, JUP, ARHGAP25, LPAR6, and PRDX1 genes, and serine/threonine kinases (STK24, STK26). Upregulation of LPAR6 showed trend towards MRD positive status (Odds ratio = 0.126; 95% CI = 0.0144-1.10; p = 0.067) and increased expression of STK26 significantly correlated with higher RFS (HR = 0.231; 95% CI = 0.0506-1.052; p = 0.04). Our findings addressed the inter- and intra-patient diversity within AML LSC and potential signaling and chemoresistance-associated targets that warrant investigation in larger cohort that may guide precision medicine in the near future.

Bio-evaluation of poly(lactic-co-glycolic) acid nanoparticles loaded with radiolabelled rifampicin.

AIMS: The poly(lactic-co-glycolic) acid (PLGA) nanoparticles of tubercular drugs have been demonstrated to have a sustained release profile over 7 days. There is no information on the location or mode of release of these nanoparticles in living systems. Therefore, we have planned the study to explore the pharmacokinetics and biodistribution of PLGA rifampicin nanoparticles in healthy human volunteers. We aim to study the distribution pattern of PLGA-loaded nano-formulation of radiolabelled rifampicin in humans. METHODS: Rifampicin was labelled with 99m Tc by indirect method and nanoparticles were prepared by a single emulsion evaporation method. To investigate the pharmacokinetics and biodistribution of nanoparticles, a single dose of 450 mg of rifampicin was administered orally to healthy human volunteers divided into two different groups. RESULTS: Following a single oral dosage of the rifampicin nanoformulation, the pharmacokinetic (PK) parameters were significantly different between the nanoparticle and conventional groups: area under the concentration-time curve (AUC = 113.8 vs. 58.6; P < .001), mean residence time (MRT = 16.2 vs. 5.8; P < .01) and elimination rate constant (Ke = 0.04 vs. 0.10; P < .05). Also, Single-photon emission computed tomography/computed tomography (SPECT/CT) images revealed biodistribution of nanoparticles in the distal portions of the intestine, which is consistent with our dosimetry analysis. CONCLUSIONS: Significant difference in PK parameters and biodistribution of nanoparticles in spleen and lymph nodes with maximum deposition were observed in the large intestine. The nanoparticle distribution pattern may be advantageous for the treatment of intestinal or lymph node tuberculosis (TB) and has the potential to result in a lower dose of rifampicin nanoformulation for the treatment of pulmonary TB.

Killer cell immunoglobulin-like receptor three domains long cytoplasmic tail 1 gene *007 may modulate disease progression of human immunodeficiency virus-1 infection in the Japanese population.

One of the KIR allele, KIR3DL1*007, was associated with the progression to acquired immunodeficiency syndrome and not with the susceptibility to HIV-1 infection in the Japanese and Indian populations, implying that KIR3DL1*007-positive NK cells might eliminate HIV-infected cells less effectively than NK cells bearing the other KIR3DL1 alleles or KIR3DS1 alleles.

Effect of Dietary Geranylgeraniol and Green Tea Polyphenols on Glucose Homeostasis, Bone Turnover Biomarkers, and Bone Microstructure in Obese Mice.

Previously, we demonstrated that the administration of either geranylgeraniol (GGOH) or green tea polyphenols (GTP) improved bone health. This study examined the combined effects of GGOH and GTP on glucose homeostasis in addition to bone remodeling in obese mice. We hypothesized that GGOH and GTP would have an additive or synergistic effect on improving glucose homeostasis and bone remodeling possibly in part via suppression of proinflammatory cytokines. Forty-eight male C57BL/6J mice were assigned to a high-fat diet (control), HFD + 400 mg GGOH/kg diet (GG), HFD + 0.5% GTP water (TP), or HFD + GGOH + GTP (GGTP) diet for 14 weeks. Results demonstrated that GTP supplementation improved glucose tolerance in obese mice. Neither GGOH nor GTP affected pancreas insulin or bone formation procollagen type I intact N-terminal, bone volume at the lumbar vertebrae, or bone parameters at the trabecular bone and cortical bone of the femur. There was an interactive effect for serum bone resorption collagen type 1 cross-linked C-telopeptide concentrations, resulting in no-GGOH and no-GTP groups having the highest values. GGOH increased trabecular number and decreased trabecular separation at the lumbar vertebrae. GTP increased trabecular thickness at lumbar vertebrae. The GG group produced the greatest connectivity density and the lowest structure model index. Only GTP, not GGOH, decreased adipokines concentrations (resistin, leptin, monocyte chemoattractant protein-1, and interleukin-6). In an obese male mouse model, individual GGOH and GTP supplementation improved glucose homeostasis, serum CTX, and trabecular microstructure of LV-4. However, the combined GGOH and GTP supplementation compromises such osteoprotective effects on serum CTX and trabecular bone of obese mice.

Transcortical resection of a giant bilobed falcine meningioma.

INTRODUCTION: Falcine meningiomas present significant surgical challenges because they often involve the falx bilaterally, are concealed by a significant amount of normal brain parenchyma and are frequently deep in location and in close proximity to the anterior cerebral arteries. Many prefer the interhemispheric approach for these lesions, but this operative corridor is not without risk as venous infarctions and cortical injury can occur. CLINICAL PRESENTATION: We present an alternative technique utilizing a transcortical approach to resect a giant, bilobed falcine meningioma in a 68-year-old female who presented with progressive abulia, urinary incontinence, and bilateral lower extremity weakness over 2 years. A unilateral right frontal craniotomy and a corticectomy through the right superior frontal gyrus was used to safely resect the entire tumor. The patient tolerated the procedure well and was discharged home without issue. Pathology demonstrated that the lesion was an atypical meningioma and she subsequently received adjuvant fractionated radiotherapy. At 2-year follow-up, she has no neurologic deficits, never developed any postoperative seizures and has not had any evidence of tumor recurrence. CONCLUSION: The transcortical approach can be used as a safe alternative for resecting falcine meningiomas without adding significant undue risk to the patient.

Mouse Sertoli Cells Inhibit Humoral-Based Immunity.

Transplantation is used to treat many different diseases; however, without the use of immunosuppressants, which can be toxic to the patient, grafted tissue is rejected by the immune system. Humoral immune responses, particularly antibodies and complement, are significant components in rejection. Remarkably, Sertoli cells (SCs), immunoregulatory testicular cells, survive long-term after transplantation without immunosuppression. The objective of this study was to assess SC regulation of these humoral-based immune factors. Mouse SCs survived in vitro human complement (model of robust complement-mediated rejection) and survived in vivo as allografts with little-to-no antibody or complement fragment deposition. Microarray data and ELISA analyses identified at least 14 complement inhibitory proteins expressed by mouse SCs, which inhibit complement at multiple points. Interestingly, a mouse SC line (MSC-1), which was rejected by day 20 post transplantation, also survived in vitro human complement, showed limited deposition of antibodies and complement, and expressed complement inhibitors. Together this suggests that SC inhibition of complement-mediated killing is an important component of SC immune regulation. However, other mechanisms of SC immune modulation are also likely involved in SC graft survival. Identifying the mechanisms that SCs use to achieve extended survival as allografts could be utilized to improve graft survival.

Immunoregulatory Sertoli Cell Allografts Engineered to Express Human Insulin Survive Humoral-Mediated Rejection.

An effective treatment and possible cure for type 1 diabetes is transplantation of pancreatic islets. Unfortunately, transplanted islets are rejected by the immune system with humoral-mediated responses being an important part of rejection. Sertoli cells (SC), an immune regulatory cell shown to survive as allografts long-term without immunosuppressants, have the potential to be used as a cell-based gene therapy vehicle to deliver endogenous insulin-a possible alternative to islets. Previously, we transduced a mouse SC line to produce human insulin. After transplantation into diabetic mice, these cells consistently produced low levels of insulin with graft survival of 75% at 50 days post-transplantation. The object of this study was to assess humoral immune regulation by these engineered SC. Both nontransduced and transduced SC survived exposure to human serum with complement in vitro. Analysis of allografts in vivo at 20 and 50 days post-transplantation revealed that despite IgG antibody detection, complement factor deposition was low and grafts survived through 50 days post-transplantation. Furthermore, the transduced SC secreted elevated levels of the complement inhibitor C1q binding protein. Overall, this suggests SC genetically engineered to express insulin maintain their ability to prevent complement-mediated killing. Since inhibiting complement-mediated rejection is important for graft survival, further studies of how SC modifies the immune response could be utilized to advance the use of genetically engineered SC or to prolong islet allograft survival to improve the treatment of diabetes.

Impact of C1q fixing donor-specific antibodies on renal transplant outcome.

Not all anti-HLA donor-specific antibodies (HLA-DSAs) are detrimental to renal allograft. In this context, the C1q complement activating ability of antibodies appears to be an important parameter to distinguish clinically inert versus detrimental DSAs. We evaluated sera of 206 consecutive primary live donor renal transplant recipients before transplant and at post-operative day 7, 30, 90, 180 and at the time of graft dysfunction for quantifying HLA-DSAs using single antigen bead assay on a Luminex platform. Patients positive for these antibodies with an MFI >500 were further screened for C1q fixing nature of DSA. Fourteen of the 18 antibody-positive patients had C1q fixing DSA with MFI value >5000. Only 4 antibody-positive patients did not have C1q fixing DSA. The MFI values of DSA detected by C1q assay were generally higher at least by 25% than those detected by the conventional IgG-SAB assay. Twelve of the 14 patients (85.71%) with C1q+ DSA developed antibody-mediated rejection during the mean follow-up period of 21.43 ± 8.03 months as compared to none of the four C1q-negative DSA (85.71% vs 0%; P = .001). These results suggest deleterious effect of C1q+ DSA vis-à-vis C1q-negative DSA on renal allograft.

Elevated pulmonary levels of Axin2 in mice exposed to herbicide 2,4-D with or without endotoxin.

2,4-Dichlorophenoxyacetic acid (2,4-D), a member of the phenoxy family of herbicides is commonly used in agriculture for controlling broadleaf weeds but its uncontrolled and incoherent use has been linked to incidences of lung toxicity. The present study aimed to understand the molecular mechanisms behind the 2,4-D alone or in combination with endotoxin (lipopolysaccharide [LPS]) induced pulmonary toxicity. Blood and lung samples were collected from Swiss albino mice (n = 48) following chronic exposure to high (37 mg/kg; 1/10th of LD50 ) and low (18.5 mg/kg; 1/20th of LD50 ) doses of 2,4-D alone or in combination with endotoxin (80 µg/animal). Transcriptome analysis revealed Wnt Canonical signaling as one of the top dysregulated pathways in mice lung following exposure to 2,4-D with and without endotoxin (LPS) co-exposure. Global view of differentially expressed genes showed increased messenger RNA expression of Axin2 by 0.26, 2.58, 3.14, 2.59, and 2.97 folds following exposure to LPS, high dose alone or in combination with LPS and low dose alone or in combination with LPS, respectively. The microarray data were validated using quantitative polymerase chain reaction and immunohistochemistry. Furthermore, the plasma concentration of Axin2 was elevated in the high dose group as revealed by Sandwich ELISA. The data taken together suggest a role of Axin2 to activate the Canonical Wnt signaling pathway in 2,4-D and or endotoxin-induced lung damage in mice.

The Good, the Bad and the Ugly of Testicular Immune Regulation: A Delicate Balance Between Immune Function and Immune Privilege.

The testis is one of several immune privilege sites. These sites are necessary to decrease inflammation and immune responses that could be damaging to the host. For example, inflammation in the brain, eye or placenta could result in loss of cognitive function, vision or rejection of the semi-allogeneic fetus, respectively. In the testis, immune privilege is "good" as it is necessary for protection of the developing auto-immunogenic germ cells. However, there is also a downside or "bad" part of immune privilege, where pathogens and cancers can take advantage of this privilege and persist in the testis as a sanctuary site. Even worse, the "ugly" of privilege is how re-emerging viruses, such as Ebola and Zika viruses, can establish persistence in the testes and be sexually transmitted even months after they have been cleared from the bloodstream. In this review, we will discuss the delicate balance within the testis that provides immune privilege to protect the germ cells while still allowing for immune function to fight off pathogens and tumors.

Clinical relevance of major histocompatibility complex class I chain-related molecule A (MICA) antibodies in live donor renal transplantation - Indian Experience.

Antibody-mediated rejections (AMR) in the absence of circulating anti-HLA-DSA have highlighted the role of non-HLA antibodies, particularly those directed against endothelial cells. Of these, MICA (major histocompatibility complex class I chain-related molecule A) antibodies are the most notable and important because of their potential in promoting graft rejections. Limited studies have focused on the impact of MICA donor-specific antibodies (DSA) on graft outcome as compared to those that are not donor-specific (NDSA). We evaluated pre- and post-transplant sera at POD 7, 30, 90, 180 and the time of biopsy from 206 consecutive primary live donor renal transplant recipients for anti-MICA and anti-HLA antibodies using single antigen bead assay on a Luminex platform. Recipients who developed MICA antibodies and their donors were phenotyped for MICA alleles. For the purpose of antibody analysis, patients were categorized into three major groups: biopsy-proven AMR, acute cellular rejection (ACR) and those with no rejection episodes (NRE). During the mean follow-up period of 17.37 ± 6.88 months, 16 of the 206 recipients developed AMR, while ACR was observed in only 13 cases. A quarter (25%) of the AMR cases had anti-MICA antibodies as compared to 7.7% of those experiencing ACR and 6.2% of the NRE group. Allelic typing revealed that all MICA Ab +ve AMR cases were due to the presence of donor-specific antibodies. MICA-DSA even in the absence of HLA-DSA was significantly associated with AMR but not with ACR when compared with the NRE group (P = <.01).

Effect of ursodeoxycholic acid in facilitating early hepatic clearance of radiotracer among patients undergoing 99mTc-sestamibi myocardial perfusion scintigraphy: A randomized double blind placebo controlled parallel trial.

BACKGROUND: Infra-cardiac tracer activity due to persistent hepatic activity interferes in inferior and infero-septal wall assessment during 99mTc-MIBI SPECT/CT myocardial perfusion scintigraphy (MPS) in evaluation of patients with coronary artery disease. It affects image interpretation with increased study duration. Ursodeoxycholic acid (UDCA) is known to enhance hepatic excretion of bilirubin and bile salts, though its role in enhancing the hepatic tracer clearance in facilitating cardiac imaging is not known. METHODS: This prospective, randomized double-blind, placebo controlled clinical trial of 120 patients, referred for adenosine stress or viability MPS studies were randomized 1:1 to receive either UDCA or placebo. Outcome was quantitative & qualitative improvement in imaging for better interpretation and to reduce the waiting time for scan. RESULTS: 118 participants (59 ± 11.9 years; 84 men) underwent adenosine stress MPS or viability MPS. Sixty participants had UDCA while 58 had placebo intervention. The study showed significant decrease in liver counts with improved myocardial to liver ratio at 30 and 60 minutes in adenosine stress MPS group, and marginally significant alteration in liver counts at 60 minutes in viability MPS group receiving UDCA, resulting in better images. CONCLUSION: UDCA intervention in MPS provides early and better image due to faster hepatic tracer clearance.

Systemic and local immunosuppression in patients with high-grade meningiomas.

AIM: Despite current treatments, high-grade meningiomas continue to have a poor prognosis. Immunotherapy targeting immune checkpoints, such as PD-L1, has demonstrated significant success in controlling numerous malignancies. In this study, we investigate the extent of systemic and local immunosuppression in meningiomas to assess the potential benefit of immune checkpoint inhibitors for the treatment of high-grade meningiomas. METHODS: Peripheral blood was collected from patients undergoing resection of meningiomas (WHO grade I, n = 18; grade II, n = 25; grade III, n = 10). Immunosuppressive myeloid cells (CD45+CD11b+PD-L1+), myeloid-derived suppressor cells (MDSCs) (CD11b+CD33+HLA-DRlow), and regulatory T cells (Tregs) (CD3+CD4+CD25+FoxP3+) were quantified through flow cytometry. Tissue sections from the same patients were assessed for PD-L1 expression and T cell infiltration via immunohistochemistry. RESULTS: Patients with grade III meningiomas demonstrated increased peripheral monocyte PD-L1 compared to patients with grade I/II meningiomas and healthy controls. Peripheral MDSC abundance was increased in grades II and III meningioma patients. PD-L1 staining of meningioma tissue demonstrated increased positivity in grade III meningiomas. Intratumoral PD-L1 was not associated with progression-free survival. High-grade meningiomas had increased T-cell infiltration. However, a significant proportion of these T cells were exhausted PD1+ T cells and immunosuppressive Tregs. CONCLUSIONS: Patients with meningiomas exhibit signs of peripheral immunosuppression, including increased PD-L1 on myeloid cells and elevated MDSC abundance proportional to tumor grade. Additionally, the tumors express substantial PD-L1 proportional to tumor grade. These results suggest a role for immune checkpoint inhibitors targeting the PD-L1/PD-1 pathway in combination with standard therapies for the treatment of high-grade meningiomas.

Tumor-induced peripheral immunosuppression promotes brain metastasis in patients with non-small cell lung cancer.

INTRODUCTION: Brain metastases are a significant source of morbidity and mortality for patients with lung cancer. Lung cancer can induce local and systemic immunosuppression, promoting tumor growth and dissemination. One mechanism of immunosuppression is tumor-induced expansion of programmed death-ligand 1 (PD-L1) expressing myeloid cells. Here, we investigate peripheral blood immune phenotype in NSCLC patients with or without brain metastasis. METHODS: Peripheral blood was collected from patients with lung metastatic brain tumors and pre-metastatic lung cancer. Immunosuppressive monocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) were quantified through flow cytometry. T cell reactivity was analyzed via ELISpot. Brain metastasis conditioned media was collected from tumor-derived cell cultures and analyzed for cytokines by ELISA. Naïve monocytes were stimulated with brain metastasis conditioned media to evaluate PD-L1 stimulation. RESULTS: Patients with brain metastatic lung carcinoma demonstrated increased peripheral monocyte PD-L1, MDSC abundance, and Treg percentage compared to early stage pre-metastatic patients and healthy controls. Patients with elevated peripheral monocyte PD-L1 had less reactive T cells and worse survival. Brain metastasis conditioned media stimulation increased monocyte PD-L1, and conditioned media IL-6 levels correlated with PD-L1 induction. Treatment with anti-IL-6 or anti-IL-6 receptor antibodies reduced PD-L1 expression. In summary, patients with lung cancer and brain metastases exhibit multiple markers of peripheral immunosuppression. CONCLUSIONS: The frequency of PD-L1+ myeloid cells correlated with the presence of brain metastases. Tumor-derived IL-6 was capable of inducing PD-L1+ myeloid cells in vitro, suggesting that monitoring of immunosuppressive factors in peripheral blood may identify new targets for therapeutic intervention in selected patients.

Comparative assessment of prognostic models in chronic lymphocytic leukemia: evaluation in Indian cohort.

Prognostic indices combining several clinical and laboratory parameters have been proposed for prognostication in chronic lymphocytic leukemia (CLL). Recently, international consortium on CLL proposed an international prognostic index (CLL-IPI) integrating clinical, molecular, and genetic parameters. The present study was designed to evaluate the reproducibility of CLL-IPI in Indian CLL cohort. The prognostic ability of CLL-IPI in terms of overall survival (OS) and time to first treatment (TTFT) was investigated in treatment-naive CLL patients and also compared with other existing prognostic scores. For assigning scores, clinical and laboratory details were obtained from medical records, and IGHV gene mutation status, ß2-microglobulin levels, and copy number variations were determined using c-DNA, ELISA, and multiplex ligation-dependent probe amplification (MLPA), respectively. The scores were generated as per the weighted grades assigned to each variable involved in score categorization. The predictive value of prognostic models was assessed and compared using Harrell's C-index and Akaike's information criterion (AIC). Stratification of patients according to CLL-IPI yielded significant differences in terms of OS and TTFT (p < 0.001). Comparative assessment of scores for OS suggested better performance of CLL-IPI (C = 0.64, AIC = 740) followed by Barcelona-Brno (C = 0.61, AIC = 754) and MDACC score (C = 0.59, AIC = 759). Comparison of predictive value of prognostic scores for TTFT illustrated better performance of CLL-IPI (C = 0.72, AIC = 726) followed by Barcelona-Brno (C = 0.68, AIC = 743), modified GCLLSG (C = 0.66, AIC = 744), and O-CLL1 index (C = 0.55, AIC = 773). The results suggest better performance of CLL-IPI in terms of both OS and TTFT as compared to other available scores in our cohort.

Lymphopenia predicts response to stereotactic radiosurgery in lung cancer patients with brain metastases.

BACKGROUND: Stereotactic radiosurgery (SRS) can enhance immune activation and improve disease control through stimulation of anti-tumor immunity. However, patients with cancer receiving chemotherapy are often immunosuppressed, which may impact the efficacy of SRS. Here we investigate the relationship between systemic lymphopenia and response to SRS in patients with brain-metastatic lung cancer. METHODS: We reviewed 125 patients with lung cancer brain metastases treated with SRS between January 2014 and May 2017. Complete blood counts from the time of SRS were reviewed, and lymphopenia was defined as absolute lymphocyte count < 1×109 cells/L. Kaplan-Meier survival analysis and cox proportional-hazards models were used to evaluate risks of progression and death. RESULTS: The median age was 65 years (range 43-86), with 54% female patients. Lymphopenia was present in 60 patients. In univariate analysis, lymphopenic patients had significantly shorter PFS (HR = 2.995, p < 0.0001) and OS (HR = 3.928, p < 0.0001). When accounting for age, gender, smoking history, ECOG score, surgery, and tumor histology in a multivariate model, lymphopenia remained significantly predictive of worse PFS (HR = 1.912, p = 0.002) and OS (HR = 2.257, p < 0.001). Patients who received immunotherapy within 3 months of SRS demonstrated significantly shorter PFS (HR = 3.578, p = 0.006) and OS (HR = 6.409, p = 0.001) if lymphopenic. CONCLUSIONS: Brain-metastatic lung cancer patients with lymphopenia treated with SRS had significantly worse PFS and OS. The effect of lymphopenia was even more pronounced in patients receiving immunotherapy. These data demonstrate the significant impact of deficient immunity on disease control and survival. Lymphopenic patients may benefit from interventions to improve immune function prior to SRS for brain metastases.

Left ventricular mechanical dyssynchrony assessment in long-standing type II diabetes mellitus patients with normal gated SPECT-MPI.

BACKGROUND: Assessment of left ventricular mechanical dyssynchrony (LVMD) using phase analysis of gated SPECT-MPI is well established. However, there is little information about the influence of diabetes mellitus on phase analysis. The present work was to evaluate the LVMD in longstanding type II diabetes mellitus (DM) patients with normal gated SPECT-MPI. METHODS: Retrospective analysis of 146 (86 type II diabetics for > 5 years' duration and 60 nondiabetics) consecutive patients with normal gated SPECT-MPI and adequate LVEF was done. Sixty age- and sex-matched nondiabetic served as control. LVMD was determined from the cutoff values (> mean + 2 SD) observed for phase standard deviation (PSD) and phase bandwidth (PBW) from the control subjects. Multivariate logistic regression analysis was applied to assess the correlation between various confounding factors. RESULTS: LVMD was detected in 24 (28%) diabetic patients with the pre-defined cut-off values for PSD (> 10.8) and PBW (> 35.6) derived from the controls. Hyperlipidemia, overweight/obesity, duration of DM and its long-term complications were independently associated with LVMD, with long-term complications being the highest risk factor (OR 28.00; P < .001). CONCLUSION: The evolution time of the patients with type II diabetes mellitus affects the left ventricular mechanical synchrony.

Sequence and Phylogenetic Analysis of the Untranslated Promoter Regions for HLA Class I Genes.

Polymorphisms located within the MHC have been linked to many disease outcomes by mechanisms not yet fully understood in most cases. Variants located within untranslated regions of HLA genes are involved in allele-specific expression and may therefore underlie some of these disease associations. We determined sequences extending nearly 2 kb upstream of the transcription start site for 68 alleles from 57 major lineages of classical HLA class I genes. The nucleotide diversity within this promoter segment roughly follows that seen within the coding regions, with HLA-B showing the highest (∼1.9%), followed by HLA-A (∼1.8%), and HLA-C showing the lowest diversity (∼0.9%). Despite its greater diversity, HLA-B mRNA expression levels determined in 178 European Americans do not vary in an allele- or lineage-specific manner, unlike the differential expression levels of HLA-A or HLA-C reported previously. Close proximity of promoter sequences in phylogenetic trees is roughly reflected by similarity of expression pattern for most HLA-A and -C loci. Although promoter sequence divergence might impact promoter activity, we observed no clear link between the phylogenetic structures as represented by pairwise nucleotide differences in the promoter regions with estimated differences in mRNA expression levels for the classical class I loci. Further, no pair of class I loci showed coordinated expression levels, suggesting that distinct mechanisms across loci determine their expression level under nonstimulated conditions. These data serve as a foundation for more in-depth analysis of the functional consequences of promoter region variation within the classical HLA class I loci.

Nucleic acid based risk assessment and staging for clinical practice in multiple myeloma.

The recently introduced Revised International Staging System (R-ISS) for multiple myeloma (MM) integrates albumin, ß2 microglobulin, lactate dehydrogenase (LDH) with high-risk cytogenetic aberrations (CA), i.e., t(4;14) and t(14;16) and del17p using fluorescent in situ hybridization (FISH). We evaluated utility of nucleic acid-based tests of multiplex ligation-based probe amplification (MLPA) and quantitative real-time polymerase chain reaction (qRT-PCR) to define the CA and the R-ISS categories as per this approach were evaluated for their ability to predict outcome in terms of response, progression-free (PFS), and overall survival (OS). In this study (n = 180), 17 (9.4%), 118 (65.6%), and 45 (25%) patients were assigned to R-ISS1, R-ISS2, and R-ISS3 categories with statistically significant differences in median PFS (p = 0.02) and OS (p < 0.001).On univariate analysis, serum creatinine, LDH, 17p deletion, chromosome 1q gain, and response after first induction therapy were associated with statistically significant differences (p < 0.05) in PFS and in addition, age> 65 years and use of triplet therapy with OS. On multivariate analysis, only serum creatinine, LDH, and response after first induction therapy retained significance for predicting PFS and in addition, use of triplet therapy retained significance for the OS. The proposed nucleic acid-based algorithm using qRT-PCR and MLPA for R-ISS is resource-effective in terms of small quantities of sample required; feasibility of batch processing and reduced overall cost for the total number of regions evaluated and retained the prognostic significance of R-ISS, making it suitable for clinical practice for molecular characterization of MM.