Detection of St. Louis encephalitis virus antigen in mosquitoes by capture enzyme immunoassay.

Surveillance methods that measure St. Louis encephalitis (SLE) virus activity in nature may provide forewarning of its epidemic occurrence in humans. An antigen capture enzyme immunoassay was developed to detect SLE virus in infected mosquitoes. The assay detected purified SLE viral antigen at a concentration of 62 pg/0.1 ml when antigen was incubated overnight; 250 pg/0.1 ml was detected in a single-day assay (antigen incubated for 3 h). The assay detected 67.9 and 70.8% of laboratory-prepared pools of infected mosquitoes after 3 h and overnight incubation, respectively. The sensitivity of the procedure was 90.5% in identifying pools with infectious titers greater than dex 3.0. The specificity of the assay was controlled by retesting positive pools preincubated with SLE virus and normal antibodies, which led to a diminution of signal in the pools containing viral antigen. The procedure was suitably specific in discriminating between SLE and related flaviviruses, detecting only high infectious doses of heterologous antigens.     

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.     

Improvement of Schwann cell attachment and proliferation on modified hyaluronic acid strands by polylysine

Hyaluronic acid (HyA) has the intrinsic ability to promote cell proliferation and reduce scar formation. However, the clinical use of HyA has so far been limited because of its water solubility and nonadhesive characteristics. Increasing interest in HyA as a clinically useful biomaterial has prompted our study of altering HyA's physical properties to render it a potential component of nerve grafts. In this study, strands of HyA were cross-linked by glutaraldehyde (Glut), coated with polylysine, and then inoculated with Schwann cells (SCs). Results in vivo and in vitro demonstrated that cross-linked HyA strands were water insoluble and thus less biodegradable. Poly-D-lysine-resurfaced strands showed significant SC attachment of 350-400 cells/mm(2), compared to uncoated controls (0-10 cells/mm(2), p < 0.01). Fibroblast control groups showed an attachment of 40-100 cells/mm(2) on coated strands. Immunostaining for proliferating cells showed SCs as and fibroblasts as +. Cells neither adhered to nor proliferated on the modified HyA strands that were not resurfaced. The results suggest that polylysine promotes SC attachment and proliferation to glutaraldehyde-cross-linked HyA strands, the product being a three-dimensional composite with low solubility that may have potential application in nerve grafts.     

Alterations of plasma antioxidants and mitochondrial DNA mutation in hair follicles of smokers

The effects of long-term smoking on mitochondrial DNA (mtDNA) deletions in hair follicles were investigated in subjects with different antioxidant capacity. Twenty-two male smokers with a smoking index of greater than 5 pack-years and without any known systemic diseases were recruited for this study. Forty healthy nonsmoking males were included as controls. We found that the concentrations of ascorbate and alpha-tocopherol and the activities of glutathione S-transferase (GST) and glutathione peroxidase in blood plasma were significantly decreased in smokers. The levels of glutathione and protein thiols in whole blood and the incidence of a 4,977 bp deletion of mtDNA (dmtDNA) in hair follicles were significantly increased in smokers. A significantly higher incidence of the 4,977 bp dmtDNA was found in smokers with plasma GST activity less than 5.66 U/l (OR = 7.2, P = 0.020). Using multiple covariate ANOVA and logistic regression, we found that age and low plasma GST activity were the only two risk factors for the 4,977 bp dmtDNA. These results suggest that smoking depletes antioxidants and causes mtDNA deletions and that plasma GST may play an important role in the preservation of the mitochondrial genome in tissue cells of smokers.     

Interferon in cerebrospinal fluid

Summary Interferon (IFN) was measured in serum and cerebrospinal fluid (CSF) of dogs after experimental (intranasal) infection with different strains of virulent canine distemper virus (CDV). Viral strains employed produce neurological changes in dogs that range from acute inflammatory to subacute, delayed demyelinating encephalomyelitis. With few exceptions, first appearance of serum-IFN correlated with the first elevated body temperature 4 days post-infection (p.i.). By 16 days p.i. IFN had disappeared from the serum of all infected dogs. In contrast, IFN was constantly detectable in CSF in dogs with CDV infection of the central nervous system (CNS). It was first detected 5 days p.i., was continuously detectable during the variable preclinical phase and into the period when signs of acute or delayed encephalomyelitis were evident. Dogs from which CDV would be retrieved from CNS tissue at necropsy always had CSF-IFN (up to 56 days p.i.). In contrast, dogs that recovered from infection, substantiated at necropsy by minimal, resolving CNS lesions and non-detectable virus, had IFN in CSF demonstrable for only a brief post-inoculation period. CSF-IFN appears to be a valid marker for CDV persistence in the canine CNS and may have broader applications.With 3 Figures     

Ovarian stimulation by clomiphene citrate and hMG in combination with cetrorelix acetate for ICSI cycles

BACKGROUND: The introduction of GnRH antagonists such as cetrorelix acetate has made possible the simplification of ovarian stimulation. However, the most effective protocol for their administration has not yet been clearly defined. METHODS: Forty women with male-factor infertility undergoing 40 ICSI cycles were included in the study. Clomiphene citrate at 100 mg a day was given from cycle day 3 through day 7. hMG at 150 IU was given on cycle days 4, 6 and 8, and was adjusted from day 9 according to the follicular and hormone responses. Cetrorelix acetate at 2.5 mg was administered when the leading follicle reached 14 mm. The remaining 0.5 mg was divided into two 0.25 mg injections for possible later use. Serum FSH, LH, estradiol and progesterone levels were measured daily from the day of cetrorelix acetate injection until hCG was given. RESULTS: Serum LH level was suppressed effectively for 4 days. Four patients (10%) needed one or two additional injections of 0.25 mg cetrorelix acetate. No premature LH surge was detected in any of the women treated. Sixteen women became pregnant (40%), of which 14 pregnancies (35%) were ongoing at the time of writing. CONCLUSIONS: This study demonstrates that this new protocol is feasible for couples with male-factor infertility undergoing ICSI.     

Bilateral simultaneous tubal sextuplets: pregnancy after in-vitro fertilization--embryo transfer following salpingectomy

The presence of a damaged tube has been suggested in recent studies to have a negative effect on in-vitro fertilization (IVF) outcome. Performing bilateral salpingectomy prior to IVF to maximize pregnancy rates may also result in unnecessary surgery. This case is also an example of the occurrence of interstitial pregnancy after salpingectomy. This unusual type of ectopic pregnancy must be kept in mind when evaluating a patient suspected of a possible early abnormal gestation after assisted reproductive technolologies.      

Human anti-JC virus serum reacts with native but not denatured JC virus major capsid protein VP1

The immunoreactivity of human anti-JC virus (JCV) serum against the major capsid protein VP1 of JCV was analyzed by Western blot, dot blot, and hemagglutination inhibition (HAI) assays. JCV-positive human serum reacted with native but not denatured JCV major capsid protein VP1, as demonstrated by dot blot and Western blot. Rabbit antiserum raised against native JCV capsid had immunoreactivities similar to those of human anti-JCV serum. These results indicate that the antigenecity of native and denatured JCV VP1 is different. In addition, both JCV-positive human serum and rabbit antiserum raised against native JCV capsid protein inhibited the hemagglutination activity of JCV capsid particles. In contrast, rabbit antiserum raised against denatured JCV VP1 did not inhibit hemagglutination. These findings reveal that denaturation may alter the antigenic epitopes of JCV VP1. Therefore, keeping the JCV capsid protein native appears to be essential for serological or other immunological analyses of the virus.