The ratio of 2nd to 4th digit length: a new predictor of disease predisposition?

The ratio between the length of the 2nd and 4th digits is: (a) fixed in utero; (b) lower in men than in women; (c) negatively related to testosterone and sperm counts; and (d) positively related to oestrogen concentrations. Prenatal levels of testosterone and oestrogen have been implicated in infertility, autism, dyslexia, migraine, stammering, immune dysfunction, myocardial infarction and breast cancer. We suggest that 2D:4D ratio is predictive of these diseases and may be used in diagnosis, prognosis and in early life-style interventions which may delay the onset of disease or facilitate its early detection.     

Round spermatids from infertile men exhibit decreased protamine-1 and -2 mRNA

During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Since patients undergoing testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) reveal low fertilization rates, whether the outcome of ICSI could be related to the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was investigated. Applying in-situ hybridization, 55 testicular biopsies from men undergoing TESE/ICSI were investigated. The percentage of PRM1-mRNA and PRM2-mRNA positive spermatids was significantly (P < 0.0001) decreased in men with at least qualitatively normal spermatogenesis (PRM1-mRNA: 58.4 +/- 13.8%; PRM2-mRNA: 56.4 +/- 11.3%) and impaired spermatogenesis (PRM1-mRNA: 32.6 +/- 10.8%; PRM2-mRNA: 31.7 +/- 11.1%) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (PRM1-mRNA: 79.9 +/- 4.6%; PRM2-mRNA: 78.1 +/- 5.7%). A positive correlation (r(PRM1) = 0.733; r(PRM2) = 0.784; P < 0.001) was demonstrated between the score and the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. While successful fertilization was neither related to the score, nor to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, a significant (P < 0.05) relationship was demonstrated between successful fertilization and the PRM1-mRNA to PRM2-mRNA ratio. Therefore, the PRM1-mRNA to PRM2-mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI.     

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species

Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.     

Shiga toxin-producing Escherichia coli isolates from cases of human disease show enhanced adherence to intestinal epithelial (Henle 407) cells.

Shiga toxin-producing Escherichia coli (STEC) strains are a diverse group of organisms which are known to cause diarrhea and hemorrhagic colitis in humans. We have recently described a large food-borne outbreak of STEC disease caused by contaminated semidry fermented sausage (A. W. Paton, R. Ratcliff, R. M. Doyle, J. Seymour-Murray, D. Davos, J. A. Lanser, and J. C. Paton, J. Clin. Microbiol. 34:1622-1627, 1996). STEC strains belonging to several O serotypes were isolated from the contaminated food source, but of these, only a subset were isolated from patients with diarrhea or hemolytic-uremic syndrome (HUS). In the present study, we characterized these STEC isolates with respect to the presence of putative virulence-associated genes and the capacity to adhere to a human intestinal epithelial cell line (Henle 407). The O111:H- STEC strain 95NR1 (isolated from one of the outbreak HUS patients) was shown to adhere to Henle 407 cells in a dose-dependent, mannose-resistant fashion. Microscopic examination revealed a diffuse pattern of adherence for this as well as several other STEC strains. Interestingly, the adherence of STEC strains from HUS cases (both outbreak related and sporadic) was significantly greater than that of STEC strains found in the contaminated food source but not found in any patients. These studies support the hypothesis that an enhanced capacity to adhere to intestinal cells is one of the factors which distinguishes human-virulent STEC strains from those of lesser clinical significance.     

Ocular measurements in fetal alcohol spectrum disorders

Fetal alcohol spectrum disorders (FASD) describe a range of physical, behavioral, and neurologic deficits in individuals exposed to alcohol prenatally. Reduced palpebral fissure length is one of the cardinal facial features of FASD. However, other ocular measurements have not been studied extensively in FASD. Using the Fetal Alcohol Syndrome Epidemiologic Research (FASER) database, we investigated how inner canthal distance (ICD), interpupillary distance (IPD), and outer canthal distance (OCD) centiles differed between FASD and non‐FASD individuals. We compared ocular measurement centiles in children with FASD to non‐FASD individuals and observed reductions in all three centiles for ICD, IPD, and OCD. However, when our non‐FASD children who had various forms of growth deficiency (microcephaly, short‐stature, or underweight) were compared to controls, we did not observe a similar reduction in ocular measurements. This suggests that reductions in ocular measurements are a direct effect of alcohol on ocular development independent of its effect on growth parameters, which is consistent with animal models showing a negative effect of alcohol on developing neural crest cells. Interpupillary distance centile appeared to be the most significantly reduced ocular measure we evaluated, suggesting it may be a useful measure to be considered in the diagnosis of FASD.     

Production of immunoglobulin A protease by Streptococcus pneumoniae from animals.

Human isolates of Streptococcus pneumoniae tested by traditional immunochemical methods produce a protease that cleaves human immunoglobulin A1 (IgA1) into Fab and Fc fragments. The protease may be an important virulence factor, but studies of its pathogenetic significance have been hampered by lack of a suitable animal model. Since S. pneumoniae is a respiratory pathogen for several species of animals, we sought to determine whether isolates of this organism from animals with pneumococcal infection, including fatal diplococcal pneumonia, produced an IgA protease. Isolates from six animal species including the mouse, rat, dog, guinea pig, rhesus monkey, and chimpanzee were tested for protease activity against IgA preparations from the mouse, rat, dog, guinea pig, rabbit, rhesus and cynomolgus monkeys, gorilla, and human. Cleavage of IgA was demonstrated by the appearance of Fc fragments in Western blots (immunoblots) treated with specific antisera. All these isolates except that from the guinea pig produced a protease that cleaved IgA of human, rhesus monkey, and gorilla origin. Cleavage was inhibited by 5 mM EDTA. IgA cleavage from the other species could not be demonstrated. Although S. pneumoniae can colonize the respiratory tracts of several animal species, it is a significant pathogen principally of humans and some other primates. Our data suggest that some species of nonhuman primates including the rhesus monkey could be suitable for experimental studies on the significance of IgA protease in the pathogenesis of pneumococcal disease.     

A role for the basal ganglia in nicotinic modulation of the blink reflex

Summary In humans and rats we found that nicotine transiently modifies the blink reflex. For blinks elicited by stimulation of the supraorbital branch of the trigeminal nerve, nicotine decreased the magnitude of the orbicularis oculi electromyogram (OOemg) and increased the latency of only the long-latency (R2) component. For blinks elicited by electrical stimulation of the cornea, nicotine decreased the magnitude and increased the latency of the single component of OOemg response. Since nicotine modified only one component of the supraorbitally elicited blink reflex, nicotine must act primarily on the central nervous system rather than at the muscle. The effects of nicotine could be caused by direct action on lower brainstem interneurons or indirectly by modulating descending systems impinging on blink interneurons. Since precollicular decerebration eliminated nicotine's effects on the blink reflex, nicotine must act through descending systems. Three lines of evidence suggest that nicotine affects the blink reflex through the basal ganglia by causing dopamine release in the striatum. First, stimulation of the substantia nigra mimicked the effects of nicotine on the blink reflex. Second, haloperidol, a dopamine (D₂) receptor antagonist, blocked the effect of nicotine on the blink reflex. Third, apomorphine, a D₂ receptor agonist, mimicked the effects of nicotine on the blink reflex.     

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.