New series of fused pyrazolopyridines: Synthesis, molecular modeling, antimicrobial, antiquorum-sensing and antitumor activities.

New series of fused pyrazolopyridines were prepared and assessed for antimicrobial, antiquorum-sensing and antitumor activities. Antimicrobial evaluation toward selected Gram-positive bacteria, Gram-negative bacteria and fungi indicated that 5-phenylpyrazolopyridotriazinone 4a has good and broad-spectrum antimicrobial activity. In addition, 5-(4-chlorophenyl)pyrazolopyridotriazinone 4b and 5-(4-(dimethylamino)phenyl)pyrazolopyridotriazinone 4c exhibited good activity against the selected Gram-positive bacteria and A. fumigatus, whereas 5-amino-4-phenylpyrazolopyridopyrimidine 6a demonstrated good activity against B. cereus and P. aeruginosa. Furthermore, 6-amino-5-imino-4-phenylpyrazolopyridopyrimidine 7a and 6-amino-4-(4-chlorophenyl)-5-iminopyrazolopyridopyrimidine 7b demonstrated promising activity against the tested Gram-negative bacteria and fungi, and moderate activity against Gram-positive bacteria. Antiquorum-sensing screening over C. violaceum illustrated that 4a, 6a and 7a-c have strong activity. In vitro antiproliferative assessment of the new derivatives against HepG2, HCT-116 and MCF-7 cancer cells revealed that 7a is the most active analog against all tested cell lines. Likewise, 3,7-dimethyl-4-phenylpyrazolopyridopyrimidinone 2a and 6-amino-4-(4-chlorophenyl)-5-iminopyrazolopyridopyrimidine 7b manifested strong activity against all examined cell lines. In vivo antitumor testing of 2a, 7a and 7b against EAC cells in mice indicated that 7a has the highest activity. Cytotoxicity toward WI38 and WISH normal cells was also assessed and results assured that all of the investigated analogs have lower cytotoxicity than doxorubicin. DNA-binding affinity and topoisomerase IIß inhibitory activity were evaluated, and results revealed that 5b, 7a and 7b bind strongly to DNA; in addition, 2a, 4a, 7a and 7b manifested higher topoisomerase IIß inhibitory activity than that of doxorubicin. Analogs 5b, 7a and 7b were docked into topoisomerase IIß, and results indicated that 7a and 7b have the highest binding affinity toward topoisomerase IIß. In silico simulation studies referred that most of the new analogs comply with the optimum needs for good oral absorption. Also, computational carcinogenicity evaluation was predicted.

Extended-spectrum beta-lactamase producing Klebsiella pneumoniae in neonatal intensive care unit.

OBJECTIVES: Extended-spectrum beta-lactamase producing (ESBL) Klebsiella pneumoniae is an important cause of nosocomial infections in neonatal intensive care units (NICUs). Our objectives were to determine (1) the incidence of ESBL K. pneumoniae in our NICU, (2) the frequency of SHV-1 and SHV-2 gene acquisition among ESBL K. pneumoniae isolates, (3) the risk factors associated with ESBL K. pneumoniae infection and (4) the clinical outcomes of infected infants. STUDY DESIGN: We conducted a prospective surveillance study in our NICU over a period of 1 year on all neonates admitted without evidence of early sepsis. We collected specimens from blood, urine, cerebrospinal fluid, swabs from wounds and throat and endotracheal tube aspirates of infants whenever sepsis was suspected. Bacterial isolates were identified via clinical morphology, Gram stain and standard biochemical tests. Antimicrobial susceptibility was determined by disc diffusion method, and phenotypic confirmation of ESBL production was done by the double-disc synergy test and Etest. Genetic detection of SHV-1 and SHV-2 genes in ESBL K. pneumoniae isolates was done by polymerase chain reaction (PCR) and restriction fragment length polymorphisms. Risk factors associated with ESBL K. pneumoniae infection were analysed by both univariate and multiple logistic regression methods. RESULTS: A total of 980 cultures were obtained from 380 neonates, and 372 screening cultures were collected from the environment. K. pneumoniae was cultured from 27 (7%) infants (3.8/1000 patient-days); of them, 18 (67%) were ESBL producers. PCR amplicons revealed the presence of SHV-2 in all 18 isolates (100%), and SHV-1 gene in 8 isolates (44%). Independent risk factors for ESBL K. pneumoniae infection were mechanical ventilation (OR: 4.2, confidence interval (CI): 1.6-11.0); birth weight <1500 g (OR: 3.2, CI: 1.2-8.3) ); duration of hospitalization >15 days (OR: 4.1, CI: 1.2-14.4); total parenteral nutrition (OR: 4.9, CI: 1.1-21.7); and previous use of oxyimino-antibiotics (OR: 4.9, CI: 1.1-21.5). ESBL was associated with higher mortality (RR=3.1, CI: 1.04-9.1) and prolonged hospitalization in those who survived (OR=3.8 CI: 1.02-11.2). Environmental cultures (n=372) had ESBL K. pneumoniae in nine isolates: four from suction tubes, two from the incubators and three from the hands of care givers. CONCLUSION: ESBL K. pneumoniae is a significant source for mortality and morbidity in infants admitted to NICU. Use of oxyimino-antibiotics is a significant risk factor for infection. The clinical significance for the SHV-1 and SHV-2 genes should be further explored.

5-Lipoxygenase and leukotriene A4 hydrolase expression in primary nephrotic syndrome.

Gene expression of 5-lipoxygenase (5-LO) and leukotriene A(4) (LTA(4)) hydrolase was analyzed in the peripheral blood of 48 children with active primary nephrotic syndrome (PNS) (group I), 27 children with PNS in remission (group II), and 20 controls. Group I included 34 patients with steroid-sensitive PNS (SSNS) and 14 patients with steroid-resistant PNS (SRNS). Total RNA purified from peripheral blood mononuclear (PBMN) cells was reverse transcribed into cDNA and amplified with specific primers in the polymerase chain reaction. All group I patients and none of the controls expressed 5-LO and LTA(4 )hydrolase. Of group II children, 22.2% expressed 5-LO, while 51.9% expressed LTA(4 )hydrolase. Among group I patients there was a significant positive correlation between the degree of proteinuria and the expression of 5-LO ( r=0.27, P=0.03) and LTA(4 )hydrolase ( r=0.44, P=0.001). There was no difference in the degree of expression of both enzymes between SSNS and SRNS patients. In conclusion, leukotrienes may play a role in the pathogenesis of PNS in children, but they do not participate in the response of these patients to steroids.

IL-10 gene expression as a marker for detection of protective immunity levels with irradiated cercarial vaccine against experimental Schistosoma mansoni infection.

Cercariae were obtained in a large number from the maintained life cycle of S. mansoni. They were attenuated at different doses (20 Kr, 50 Kr, 60 Kr, 70 Kr and 80 Kr) of gamma radiation. Laboratory bred Swiss Albino mice were classified into 7 groups. Five groups were immunized with +/-500 S. mansoni cercariae. Two groups were used as positive and negative controls. All animals were sacrificed after 8 weeks. Spleen cell proliferative responses to Phytohaemagglutinin (PHA) were assessed in all groups. IL-10 was measured by ELISA in serum and splenic cells secretion in-vitro. RNA extracted from freshly isolated liver cells was analyzed for detection of mRNA of IL-10 by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed augmentation of proliferative cell from the spleen in all vaccinated groups except with 80 Kr, irradiated cercariae group. The highest percentage of lymphocytes transformation was recorded among the mice immunized with 60 Kr, irradiated cercariae. After challenge, splenic responses in all groups declined progressively to the control level. IL-10 secretion from spleen cells of all vaccinated groups increased after challenge with the least level in 60 Kr, immunized challenged group. IL-10 mRNA expression was higher among 60 Kr, immunized with irradiated cercariae mice group than 70 Kr, one, but with no expression among 80 Kr, cercariae immunized ones.

Immune modulatory potentials of antineoplaston A-10 in breast cancer patients.

Antineoplastons are naturally occurring cytodifferentiating agents. Chemically, they are medium and small sized peptides, amino acid derivatives and organic acids, which exist in blood, tissues and urine. Antineoplaston A-10 (3-phenylacetylamino-2,6-piperidinedione) is the first chemically identified antineoplaston. Previously we have shown a strong inverse association of urinary antineoplaston A-10 with breast cancer. This study is designed to evaluate neutrophil apoptosis in patients with breast cancer at time of diagnosis and to correlate urinary antineoplaston A-10 levels with neutrophil apoptosis and to describe the direct effect of A-10 in vitro on neutrophil apoptosis in breast cancer patients. The participants were patients with a histologically confirmed diagnosis of breast cancer. Only those cases without previous treatment for breast cancer were included. Neutrophil apoptosis was assessed in breast cancer patients both morphologically and by DNA fragmentation and studied relative to healthy controls. Antineoplaston A-10 was measured using high performance liquid chromatography in urine samples collected from the patients. Urine samples from normal women served as controls. Direct effect of antineoplaston A-10 on neutrophil apoptosis was tested in vitro after adding A-10 at a concentration of 10 ng/ml to the cellular suspensions of breast cancer patients. Non-treated samples served as controls. Significantly higher neutrophil apoptosis levels were detected among patients with breast cancer with a P value <0.001. Urinary antineoplaston A-10 level is significantly negatively correlated with high apoptosis levels (P<0.0001). In vitro, antineoplaston A-10 was found to inhibit significantly the neutrophil apoptosis with a P value <0.0001. These findings confirm the presence of immune defects among patients with breast cancer and such results should stimulate the development of new strategies to induce and augment immunity for the treatment of breast cancer. Antineoplaston A-10 may provide rational basis for designing trials to employ its immune modulatory potentials as adjuvant therapy in breast cancer patients.

Deltamethrin-induced testicular apoptosis in rats: the protective effect of nitric oxide synthase inhibitor.

This study is the first to examine and characterize the testicular apoptosis which might be induced due to exposure of male rats to deltamethrin. Furthermore, the role which might be played by nitric oxide (NO), as well as the other reactive oxygen species (ROS) in controlling this testicular apoptosis was assessed. Apoptosis was evaluated by DNA fragmentation detected by agarose gel electrophoresis and cellular morphology on testicular tissue sections. It was found that administration of deltamethrin (1 mg/kg daily for 21 days) to animals resulted in characteristic DNA migration patterns (laddering), thereby providing evidence that apoptosis is the major mechanism of cell death in the testicular tissues. In addition, histopathological examination of testicular tissue sections showed that apoptosis was confined to the basal germ cells, primary and secondary spermatocytes. These changes, in addition to the appearance of Sertoli cell vacuoles in deltamethrin-intoxicated animals, indicates the suppression of spermatogenesis. At the same time, the plasma levels of both NO and lipid peroxides measured as malondialdehyde (MDA) were found to be significantly increased in deltamethrin-treated animals. Administration of NO synthase (NOS) inhibitors such as N(G)-nitro monomethyl L-arginine hydrochloride (L-NMMA, 1 mg/kg) to rats 2 h before exposure to deltamethrin was effective in the reduction of the typically testicular apoptotic DNA fragmentation pattern and the associated histopathological changes. These findings may suggest that deltamethrin-induced testicular apoptosis is mediated by NO. Therefore, the pharmacological manipulation of apoptosis by selective NOS inhibitors such as L-NMMA may offer new possibilities for the control of deltamethrin-induced testicular dysfunction and infertility in the future.

Susceptibility to and severity of rheumatoid arthritis in multicase families.

Fifteen sibships, each having two or more siblings affected by classical or definite RA were studied. They comprised 31 patients with RA (all in remission) and 21 normal siblings. The total severity index of RA was assessed by clinical and radiological indices. For all the patients the following investigations were carried out: (1) HLA antigens determination for nine antigens at A locus and 15 at B locus and 6 at DR locus; (2) rheumatoid factor. We found: (1) RA disease is genetically controlled and the responsible genes are linked to the HLA system; (2) association between seropositivity and DR4 and DR4/B27 genotypes; (3) significant effect of the genotype DR4/B27 on the age of onset; (4) association between the increase in disease severity both clinical and radiological and DR4/X and DR4/B27 phenotypes. Thus the genetic control is probably composed of two types of genes: disease susceptibility genes and disease severity genes linked to DR4/X and DR4/B27 phenotypes.

Evidence of inherited susceptibility of increased streptococcal adherence to pharyngeal cells of children with rheumatic fever.

The study included the members of 15 families, each having more than one sibling affected by rheumatic fever (RF). All the rheumatic individuals showed the sequelae of rheumatic carditis, but on clinical and laboratory evidence, the disease was inactive. Thirty normal unrelated individuals, having no rheumatic first-degree relatives, were studied as controls. The following investigations were carried out for all members: (1) history and clinical examination, (2) routine investigations of diagnosis, (3) HLA typing using 9-A, 15-B, 6-DR antigens, (4) adherence of group A streptococci to pharyngeal cells, an in vitro adherence assay. There were two types of strains; five RF-associated strains and two RF-unassociated strains. Statistical and genetic analysis revealed: (1) no significant difference between adherence of RF-associated and unassociated strains amongst controls; (2) significant increased avidity for adherence of RF-associated strains amongst rheumatic siblings compared to normal siblings and controls. There was no significant difference between the three groups using RF-unassociated strains; (3) HLA-haplotype concordance and 'N' measure showed that the avidity for adherence is probably inherited; (4) lod scores for linkage suggest a dominant susceptibility gene(s) closely linked to HLA and segregating in multiplex families.

Sporadic carriers of human T-lymphotropic virus type I in northern Egypt.

Sera from 3,158 individuals living in northern Egypt were tested for the presence of antibodies against human T-lymphotropic virus type I (HTLV-I) by the newly developed particle agglutination (PA) test. Ten sera gave a positive reaction in the PA test. Eight of these sera were examined further by Western blotting and all of them gave several bands corresponding to HTLV-I structural proteins. Two of the 8 sera gave positive results in the indirect immunofluorescence test. The results indicate the presence of HTLV-I carriers in this area, although at very low incidence (0.063%).