Preclinical evaluation of group B Neisseria meningitidis and Escherichia coli K92 capsular polysaccharide-protein conjugate vaccines in juvenile rhesus monkeys.

We reported the first use of group B meningococcal conjugate vaccines in a nonhuman primate model (S. J. N. Devi, C. E. Frasch, W. Zollinger, and P. J. Snoy, p. 427-429, in J. S. Evans, S. E. Yost, M. C. J. Maiden, and I. M. Feavers, ed., Proceedings of the Ninth International Pathogenic Neisseria Conference, 1994). Three different group B Neisseria meningitidis capsular polysaccharide (B PS)-protein conjugate vaccines and an Escherichia coli K92 capsular polysaccharide-tetanus toxoid (K92-TT) conjugate vaccine are here evaluated for safety and relative immunogenicities in juvenile rhesus monkeys with or without adjuvants. Monkeys were immunized intramuscularly with either B PS-cross-reactive material 197 conjugate, B PS-outer membrane vesicle (B-OMV) conjugate, or N-propionylated B PS-outer membrane protein 3 (N-pr. B-OMP3) conjugate vaccine with or without adjuvants at weeks 0, 6, and 14. A control group of monkeys received one injection of the purified B PS alone, and another group received three injections of B PS noncovalently complexed with OMV. Antibody responses as measured by enzyme-linked immunosorbent assay varied among individual monkeys. All vaccines except B PS and the K92-TT conjugate elicited a twofold or greater increase in total B PS antibodies after one immunization. All vaccines, including the K92-TT conjugate, elicited a rise in geometric mean B PS antibody levels of ninefold or more over the preimmune levels following the third immunization. Antibodies elicited by N-pr. B-OMP3 and B-OMV conjugates were directed to the N-propionylated or to the spacer-containing B PS antigens as well as to the native B PS complexed with methylated human serum albumin. None of the vaccines caused discernible safety-related symptoms.     

Control of hair follicle cell fate by underlying mesenchyme through a CSL–Wnt5a–FoxN1 regulatory axis

Epithelial–mesenchymal interactions are key to skin morphogenesis and homeostasis. We report that maintenance of the hair follicle keratinocyte cell fate is defective in mice with mesenchymal deletion of the CSL/RBP-Jκ gene, the effector of “canonical” Notch signaling. Hair follicle reconstitution assays demonstrate that this can be attributed to an intrinsic defect of dermal papilla cells. Similar consequences on hair follicle differentiation result from deletion of Wnt5a, a specific dermal papilla signature gene that we found to be under direct Notch/CSL control in these cells. Functional rescue experiments establish Wnt5a as an essential downstream mediator of Notch–CSL signaling, impinging on expression in the keratinocyte compartment of FoxN1, a gene with a key hair follicle regulatory function. Thus, Notch/CSL signaling plays a unique function in control of hair follicle differentiation by the underlying mesenchyme, with Wnt5a signaling and FoxN1 as mediators.     

Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR

The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.     

Novel and recurrent germline LEMD3 mutations causing Buschke–Ollendorff syndrome and osteopoikilosis but not isolated melorheostosis

Mutations in the LEMD3 gene were recently incriminated in Buschke–Ollendorff syndrome (BOS) and osteopoikilosis, with or without melorheostosis. The relationship of this gene with isolated sporadic melorheostosis is less clear. We investigated LEMD3 in a two-generation BOS family showing an extremely variable expression of the disease, in a sporadic patient with skin features of BOS, and in an additional subject with isolated melorheostosis. We identified two different mutations, both resulting in a premature stop codon, in the two cases of BOS. The mutation (c.2564G>A) reported in the familial case is novel, while that observed in the sporadic case (c.1963C>T) has been previously reported in an American woman with osteopoikilosis and melorheostosis who had a family history of isolated osteopoikilosis. The search for mutations in DNA extracted from the peripheral blood, as well as skin and bone biopsies of the patient with melorheostosis failed to identify any pathogenic change. Our results further expand the LEMD3 mutation repertoire, corroborate the extreme interfamilial and intrafamilial clinical variability of LEMD3 mutations, and underline the lack of a clear phenotype–genotype correlation in BOS. The present study supports the general conclusion that LEMD3 mutations do not contribute to isolated sporadic melorheostosis. The genetic or epigenetic influences that are responsible for the development of melorheostosis require further investigation.     

Preservation of the antioxidant status in chemically-induced diabetes mellitus by melatonin

Oxidant stress is believed to be enhanced in patients with diabetes mellitus, which may lead to endothelial dysfunction and the development of atherosclerosis. In diabetes, hyperglycemia drives non-enzymatic glycation and oxidation of proteins and lipids which enhances the formation of advanced glycation end products (AGEs), which may be involved in the pathogenesis of diabetic vascular disease. The macrovascular complications of diabetes seem to be due to enhanced cellular oxidant stress by the interaction of AGEs with their receptor. It would be worthwhile to devise methods to reduce this oxidant stress. In alloxan-induced diabetic rats lipid peroxidation products were increased, while levels of nitric oxide glutathione peroxidase and superoxide dismutase were reduced. Melatonin restored these biochemical abnormalities to normalcy independent of hyperglycemia. This model can be used to study the role of oxidant stress in the development of macrovascular complications in diabetes mellitus.     

Antitumor and Radiosensitizing Effects of Withaferin a on Mouse Ehrlich Ascites Carcinoma in vivo

The antitumor and radiosensitizing effects of withaferin A (WA), a steroidal lactone from Withania somnifera, was studied on Ehrlich ascites carcinoma in vivo. The acute LD₅₀₍₁₄₎ for WA in Swiss mice was ∼ 80 mg/kg. Twenty-four hours after i.p. inoculation of 10⁶ tumor cells, WA was injected i.p. at different dose fractions (5 or 7.5 mg/kg × 8, 10 mg/kg × 5, 20 or 30 mg/kg × 2) with or without abdominal gamma irradiation (RT, 7.5 Gy) after the first drug dose. Increase in life span and tumor-free survival were studied up to 120 days. The drug inhibited tumor growth and increased survival, which was dependent on the WA dose per fraction rather than the total dose. Combination of RT with all the drug schedules increased tumor cure and tumor-free survival, the best effect seen after 2 fractions of 30 mg/kg each. In another experiment WA was given as 2 (40 mg/kg × 2), 3 (30 mg/ kg × 3) or 4 (20 mg/kg × 4) fractions at 5, 7 or 10 days after tumor inoculation with or without RT after the first drug dose. At 7 and 10 days after inoculation the drug was effective only at 40 mg/kg × 2, but with RT 30 mg/kg × 3 produced an equal effect (20% survival) on 7 day old tumors.     

Isolation and evaluation of diagnostic value of two major secreted proteins of Mycobacterium tuberculosis

Two secreted antigens of Mycobacterium tuberculosis, namely the antigen 85 complex (30/31) and 38kDa antigens, were purified from the whole culture filtrate by using two dimensional preparative electrophoresis and anion exchange chromatography, respectively. Individual components of the antigen 85 complex namely, antigen 85A, 85B and 85C, were separated using hydrophobic interaction chromatography. The humoral antibody activity to these antigens in sputum positive cases of active pulmonary tuberculosis and normal healthy volunteers was determined by enzyme linked immunosorbent assay (ELISA) and immunoblot. Recombinant 38kDa and antigen 6 were used as reference antigens for the assay. None of the healthy volunteers reacted with the 38kDa antigen, while 52% of the TB sera reacted with it. Of the three components of the antigen 85 complex, 85B gave the highest positivity of 40 per cent. The results of combination of 38kDa with antigen 6 offered better results with 76% positivity.     

Insulin-like growth factor binding protein-3 induces early apoptosis in malignant prostate cancer cells and inhibits tumor formation in vivo

BACKGROUND: Insulin-like growth factor binding protein (IGFBP-3) levels are significantly reduced in malignant prostate epithelial cells. In this study, we evaluated the role of endogenous IGFBP-3 on prostate cancer cell growth and tumorigenesis. METHODS: IGFBP-3 was re-expressed by stable transfection of human IGFBP-3 cDNA in a model of human prostate cancer, M12, a malignant subline in which IGFBP-3 levels are undetectable in comparison to the parent epithelial cell, P69. Effect of IGFBP-3 re-expression (M12-BP-3) on growth kinetics, morphology, propensity to apoptosis, and in vivo tumor formation were studied. RESULTS: M12-BP-3 cells secreted IGFBP-3 and growth arrested at a cell density that was threefold lower than control cells and this was associated with marked alteration in cell morphology. Control cells when grown in conditioned media secreted by M12-BP-3 also showed altered morphology compared to when cultured in IGFBP-3-immunodepleted conditioned media. The M12-BP-3 clones showed altered mitochondrial membrane potential, increased PARP cleavage, increase in sub-G1 peak, decreased levels of neuron specific enolase, and decreased tumor formation in athymic, nude mice. CONCLUSIONS: These data suggest that IGFBP-3 induces early apoptosis and has potential tumor suppressive effect in prostate cancer. Prostate 51: 141-152, 2002.