Establishment of an optimal hypo-osmotic swelling test by examining single spermatozoa in four different hypo-osmotic solutions

In order to find an optimal hypo-osmotic swelling test (HOST) and to identify viable sperm cells from patients with asthenozoospermia for intracytoplasmic sperm injection (ICSI), we tested single motile and non-motile spermatozoa in four hypo-osmotic solutions by micromanipulation. The four solutions were: A, H2O; B, 50 mOsm NaCl; C, 150 mOsm NaCl and D, 150 mOsm sodium citrate and fructose. Eosin Y staining was then carried out for evaluation of viability of the spermatozoa after HOST. Using motile spermatozoa, no statistical difference was found in HOST-positive spermatozoa between these four solutions. There were more viable sperm cells after HOST in solutions C and D, as noted by Eosin Y staining. After non-motile spermatozoa were incubated for 1 min in the four solutions, HOST with solution C gave the best results for identification of viable sperm cells compared to the other three solutions. When motile spermatozoa were incubated in solution C or solution D for 30 min, the result of HOST with solution C (10.8% dead spermatozoa) was superior to that of solution D (49.1% dead spermatozoa). In conclusion, the HOST protocol using 150 mOsm NaCl (solution C) for 1 min yielded the best results for selection of viable spermatozoa. This procedure should be used for selection of viable spermatozoa for ICSI in patients with 100% non-motile spermatozoa.      

Linkage analysis of the genetic determinants of T-cell IL-4 secretion, and identification of Flj20274 as a putative candidate gene

The activation-induced differentiation of naive CD4+ T cells generates functionally divergent type 1 helper T cells (Th1) or type 2 helper T cells (Th2) effector cell populations, characterized by secretion of Interferon (IFN)-gamma or Interleukin (IL)-4, respectively. Inappropriate generation of Th subsets may contribute to immune dysfunction. The decision to generate Th1/Th2 lineages is critically regulated by cytokines, such that IL-12 induces Th1 differentiation, while IL-4 induces Th2 differentiation. Genetic factors influence the pathway of Th differentiation, as displayed by the preferential generation of divergent Th populations by different inbred strains of mice. We employ two complementary genetic techniques to identify genes that regulate the default IL-4 secretion profiles of T cells from BALB/c and B6 mice. We performed a genome-wide linkage analysis of the progeny of a backcross between BALB/c and B6 mice to identify three loci, T-cell secretion of interleukin-4 (Tsi)1-3, on chromosomes 7, 19 and 15, respectively, which regulate in vitro T-cell IL-4 production. We have also employed mRNA representational difference analysis to isolate a gene, Flj20274, which is differentially expressed in T cells that secrete high levels of IL-4. Significantly, Flj20274 was mapped to the point of peak linkage within Tsi1 and is a strong candidate for Tsi1.     

CAG repeat expansion in autosomal dominant pure spastic paraplegia linked to chromosome 2p21-p24

CAG repeat expansions have been identified as the disease-causing dynamic mutations in the coding regions of genes in several dominantly inherited neurodegenerative disorders, including spinobulbar muscular atrophy, Huntington's disease, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia type 1, 2 and 6 and Machado-Joseph disease. The CAG repeat expansions are translated to elongated polyglutamine tracts and an increased size of the polyglutamine tract correlates with anticipation, the cardinal feature, seen in all these diseases. Autosomal dominant pure spastic peraplegia (ADPSP) is a degenerative disorder of the central motor system clinically characterized by slowly progressive and unremitting spasticity of the legs, hyperreflexia and Babinski's sign. Like the established CAG repeat diseases ADPSP is characterized by both inter- and intrafamilial variation and anticipation. Using the Repeat Expansion Detection (RED) method, we have analyzed 21 affected individuals from six Danish families with the disease linked to chromosome 2p21-p24. We found that 20 of 21 affected individuals showed CAG repeat expansions versus two of 21 healthy spouses, demonstrating a strongly statistically significant association between the occurrence of the repeat expansion and the disease (Fisher's test, P < 10(-5)) suggesting that a CAG repeat expansion is involved presumably as a dynamic mutation in ADPSP linked to chromosome 2p21-p24. The size of the expansion is estimated to be > or = 60 CAG repeat copies in the affected individuals. The CAG repeat expansion is very likely translated and expressed as indicated by the detection of a polyglutamine-containing protein in an ADPSP patient.      

Hyperexplexia: an inherited disorder of the startle response

A family is presented with hyperexplexia, a rare autosomal dominant neurological disorder. Affected individuals manifest flexor hypertonia and hypokinesia during infancy. Later and throughout life, the condition is characterized by exaggerated involuntary myoclonic startle reactions, which on occasion result in failing. There are also marked nocturnal myoclonic jerks. Many family members have had congenital hip dislocations and inguinal hernias. Pre- and postnatal hypertonia is proposed as the cause for these problems. The nature and location of central nervous system dysfunction in hyperexplexia was investigated using electroencephalographic and brainstem-evoked response techniques. A dysfunction of cortical inhibition of the brainstem-mediated startle response is discussed as a possible pathogenic mechanism. Accurate diagnosis of this disorder is important in order to provide appropriate counseling and to initiate effective treatment.     

Zinc deficiency and anorexia in rats: normal feeding patterns and stress induced feeding

We report the effects of zinc deficiency on normal feeding behavior in rats and the effects of zinc deficiency on stress-induced eating in rats. Zinc deficient (ZD) rats weighed significantly less than their pair-fed and ad lib controls. Zinc repletion allowed improved growth, but ZD rats never displayed catch-up growth compared to their ad lib controls. ZD rats rapidly developed a depressed food efficiency ratio which normalized with zinc supplementation. Food consumption in ZD rats was approximately one-third that of ad lib controls and water intake was also significantly reduced. Mild tail pinch was able to induce feeding in these normally anorexic ZD rats. We conclude that zinc deficiency represents an interesting model of anorexia which may enhance our understanding of appetite regulation.     

Complications common to ventricular assist device support are rare with 90 days of DeBakey VAD support in calves

The DeBakey VAD is a miniaturized, electromagnetically driven axial flow pump intended for long-term ventricular assist. Safety and performance data from six calves implanted with the complete DeBakey VAD system are reported elsewhere; here we describe complications and necropsy findings for these same six animals, all of which survived 90 days. The study was conducted according to a uniform protocol, which included anticoagulation and antibiotic prophylaxis. Clinical complications tracked included bleeding, cardiovascular abnormalities (e.g., arrhythmias, tachycardia unrelated to pain, bradycardia), hemolysis, hepatic dysfunction, renal dysfunction, thromboembolism (neurologic or peripheral), or infection. Each adverse event was retrospectively categorized with regard to severity (mild, moderate, severe) and relationship to device. Clinical findings were confirmed by necropsy. There was no evidence of systemic infection, thromboembolism, hemolysis, or renal or hepatic dysfunction in these six animals during the study period. A single adverse event was noted in each of two of the calves. Both events were considered mild according to the predefined criteria. Bleeding related to the surgical implantation procedure and requiring reoperation occurred in one animal. The other animal had evidence of a superficial infection at the exit site of the cables on the left lateral thoracic wall; the infection did not extend into the thoracic cavity. Chronic, healed small renal infarct scars were present in several animals. Mild valvular endocardiosis was observed in two calves and mild fibroelastosis was present in the endocardium at the site of the inflow cannula in three calves; however, these lesions were not considered clinically significant. No other gross or histologic abnormalities were noted at necropsy. In conclusion, calves implanted with the complete DeBakey VAD for 90 days demonstrated few complications and had no significant necropsy findings. Complications common to ventricular assist device (VAD) support (i.e., hemolysis, infection, bleeding, thromboembolism) were rare during long-term support (90 days) with the DeBakey VAD.