GRP78-targeting subtilase cytotoxin sensitizes cancer cells to photodynamic therapy.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER)-resident chaperone and a major regulator of the unfolded protein response (UPR). Accumulating evidence indicate that GRP78 is overexpressed in many cancer cell lines, and contributes to the invasion and metastasis in many human tumors. Besides, GRP78 upregulation is detected in response to different ER stress-inducing anticancer therapies, including photodynamic therapy (PDT). This study demonstrates that GRP78 mRNA and protein levels are elevated in response to PDT in various cancer cell lines. Stable overexpression of GRP78 confers resistance to PDT substantiating its cytoprotective role. Moreover, GRP78-targeting subtilase cytotoxin catalytic subunit fused with epidermal growth factor (EGF-SubA) sensitizes various cancer cells to Photofrin-mediated PDT. The combination treatment is cytotoxic to apoptosis-competent SW-900 lung cancer cells, as well as to Bax-deficient and apoptosis-resistant DU-145 prostate cancer cells. In these cells, PDT and EGF-SubA cytotoxin induce protein kinase R-like ER kinase and inositol-requiring enzyme 1 branches of UPR and also increase the level of C/EBP (CCAAT/enhancer-binding protein) homologous protein, an ER stress-associated apoptosis-promoting transcription factor. Although some apoptotic events such as disruption of mitochondrial membrane and caspase activation are detected after PDT, there is no phosphatidylserine plasma membrane externalization or DNA fragmentation, suggesting that in DU-145 cells the late apoptotic events are missing. Moreover, in SW-900 cells, EGF-SubA cytotoxin potentiates PDT-mediated cell death but attenuates PDT-induced apoptosis. In addition, the cell death cannot be reversed by caspase inhibitor z-VAD, confirming that apoptosis is not a major cell death mode triggered by the combination therapy. Moreover, no typical features of necrotic or autophagic cell death are recognized. Instead, an extensive cellular vacuolation of ER origin is observed. Altogether, these findings indicate that PDT and GRP78-targeting cytotoxin treatment can efficiently kill cancer cells independent on their apoptotic competence and triggers an atypical, non-apoptotic cell death.

Frequency of distribution of inflammatory cytokines IL-1, IL-6 and TNF-alpha gene polymorphism in patients with obstructive sleep apnea.

Obesity is one of the most commonly identified factors for the obstructive sleep apnea syndrome (OSAS). Adipose tissue is the source of many cytokines, among them there are IL-6, IL-1, and TNF-alpha. The level of inflammatory cytokines increases in people with OSAS and obesity. The aim of this study was to evaluate the distribution of genotypes in inflammatory cytokine genes in people with obesity-related OSAS. The examined group consisted of 102 person with obesity related-OSAS and 77 normal weight person without OSAS. Genotyping of DNA sequence variation was carried out by restriction enzyme (IL-1: Taq I, IL-6: Lwe I, TNF-alpha: Nco I) analysis of PCR amplified DNA. The study revealed a significant correlation between polymorphism located in the promoter region of inflammatory cytokine genes and obesity-related OSAS.

Frequency of distribution of leptin receptor gene polymorphism in obstructive sleep apnea patients.

Leptin is an adipocyte-derived hormone regulating energy homeostasis and body weight. Leptin concentration is increased in patients with the obstructive sleep apnea syndrome (OSAS). Leptin receptor (LEPR) is a single transmembrane protein belonging to the superfamily of cytokine receptors related by a structure to the hemopoietin receptor family. The aim of the present study was to evaluate the frequency of distribution of leptin receptor gene polymorphism GLN223ARG in OSAS patients compared with healthy controls. The examined group included 179 subjects: 102 OSAS patients (74 men and 28 women) and 77 non-apneic controls (39 men and 38 women). Genomic DNA was isolated with the use of a column method and genotyping of DNA sequence variation was carried out by restriction enzyme analysis of PCR-amplified DNA. The results revealed a significant correlation between the polymorphism of LEPR and OSAS. Carriers of Arg allele in homozygotic genotype Arg/Arg and heterozygotic genotype Gln/Arg were more often obese and developed OSAS than the group of carriers of homozygotic Gln/Gln genotype. This tendency was observed in the whole examined population and in the group of obese women. We also found the highest levels of total cholesterol, LDL, HDL, and triglycerides in the group of homozygotic Arg/Arg genotype carriers, lower in heterozygotic Gln/Arg genotype carriers, and the lowest in the group of persons carring homozygotic Gln/Gln genotype. The presence of Arg allel seems linked to a higher risk of obesity and higher lipid levels in OSAS patients. OSAS may have a strong genetic basis due to the effects from a variety of genes including those for leptin receptor.

Metformin-diet ameliorates coronary heart disease risk factors and facilitates resumption of regular menses in adolescents with polycystic ovary syndrome.

BACKGROUND: In 35 adolescent females (17 +/- 2 years) with polycystic ovary syndrome (PCOS), median body mass index (BMI) 30.8 kg/m2, we assessed effeicacy of metformin-diet for 1 year for reduction of weight, insulin, HOMA insulin resistance (IR), cholesterol, triglycerides, and resumption of regular menses. METHODS: Calories (26% protein, 44% carbohydrate) were targeted to 1,500-1,800/day if BMI was <25 or to 1,200-1,500/day if BMI was > or = 25, along with 2,550 mg metformin. RESULTS: Median weight fell from 82.7 to 79.1 kg (p = 0.009), insulin 16.7 to 13.3 microU/ml (p <0.0001), HOMA IR 3.41 to 2.74 (p = 0.0004), total cholesterol 164 to 151 mg/dl (p = 0.002), and triglyceride 103 to 85 mg/dl (p = 0.006). The percentage of cycles with normal menses rose from a pre-treatment mean of 22% to 74%, p < 0.0001. CONCLUSIONS: In adolescents with PCOS, metformin-diet reduces weight, insulin, IR, cholesterol, and triglycerides, and facilitates resumption of regular menses.

A highly specific and sensitive sandwich blocking ELISA based on baculovirus expressed pseudorabies virus glycoprotein B.

A direct sandwich blocking enzyme-linked immunosorbent assay (BacgB ELISA) based on the reaction between a monoclonal antibody (MAb) and a recombinant glycoprotein B (gB) of pseudorabies virus (PRV) was developed. This protein was obtained in large quantities from insect cells infected with a PRV gB recombinant baculovirus. Expression of the gB was confirmed by immunoperoxidase monolayer assay (IPMA) with gB specific MAbs. The specificity and sensitivity of the developed BacgB ELISA were evaluated and compared with two commercially available tests by using sets of sera of known PRV infection or vaccination history. For validation, 347 serum samples have been tested. The BacgB ELISA had a high sensitivity and specificity, which were comparable with those of the two commercial tests. In addition, the BacgB ELISA allows detecting anti-gB antibodies in pig serum as early as 7 days following infection. Also maternal antibodies in uninfected pig sera were detected. We conclude that the BacgB ELISA is a useful tool for the detection of as well vaccinated as infected pigs (including derivatives from gE negative vaccine strains), with the added advantage that it uses an antigen that can be produced safely and in large quantities.

Coincidence between spontaneous release of interferon and tumor necrosis factor by colostral leukocytes and the production of a colostrinine by human mammary gland after normal delivery.

We have tentatively identified colostrinines as novel cytokines produced by the mammary gland after delivery and detectable in colostrum. The primary colostrinine, the proline-rich polypeptide, was isolated from ovine colostrum in 1974. It is generally understood that the various factors present in colostrum play a pivotal role in transmitting of passive or active immunity from mother to child. We have found previously that both ovine and human colostrinines are inducers of interferon (IFN) gamma and other cytokines. In this paper, we reported that the leukocytes isolated from human colostrum donated by healthy mothers at 1-9 days after delivery, produced IFNs and tumor necrosis factors (TFNs) spontaneously. The release of IFNs and TNFs coincided with production of a colostrinine that has been isolated from the human colostrum samples and partially characterized. Our results suggest that the maximum production of colostrinine occurs 3 days after delivery. The tolerance (hyporeactivity) of the colostral leukocytes to IFN inducers and the modulation of the TNF response may be the late effects of the colostrinine release.