Affinity fractionation of lymphocytes using a monolithic cryogel

A new type of continuous, supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing specific fractionation and separation of human peripheral blood lymphocytes in a chromatographic format. The affinity adsorbent was used to design a novel cell separation strategy, which was based on the interaction of protein A from Staphylococcus aureus with cells bearing IgG antibodies on the surface. After treating lymphocytes with goat anti-human IgG(H+L), the IgG-positive B-lymphocytes were efficiently separated from T-lymphocytes. Protein A covalently coupled to epoxy activated dimethylacrylamide (DMAA) cryogel matrix specifically bound IgG-bearing B-lymphocytes through the Fc region, while non-bound T-lymphocytes passed through the column. More than 90% of the B-lymphocytes were retained in the column while the cells in the breakthrough fraction were enriched in T-lymphocytes (81%). The viability of the T-lymphocytes isolated was greater than 90%. The bound lymphocytes released by human or dog IgG recovered 60-70% of the B-cells without significantly impairing the cell viability. The technique can be applied in general to cell separation systems where IgG antibodies against specific cell surface markers are available.     

An effective regimen for early medical abortion: a report of 2000 consecutive cases

A combination of the anti-progesterone mifepristone and gemeprostprovides an effective non-surgical method for the inductionof abortion at gestations up to 63 days, achieving completeabortion rates of over 95%. We report our experience with analternate regimen, comprising a reduced dose of mifepristonein combination with vaginal misoprostol. A consecutive seriesof 2000 women requesting early medical abortion at gestationsup to 63 days was studied retrospectively. Each woman receivedmifepristone 200 mg orally, followed 36–48 h later bymisoprostol 800 µg vaginally. Of the 2000 women, 39 (2.0%)aborted completely following administration of mifepristonealone and a further 1912 experienced complete abortion followingadministration of misoprostol (a complete abortion rate of 97.5%).Surgical intervention was required in 49 women (2.5%): for incompleteabortion in 27 (1.4%), for missed abortion in seven (0.4%),for continuing pregnancy in 11 (0.6%) and to exclude ectopicpregnancy in four (0.2%). The surgical intervention rate wassignificantly higher among women at gestations 49 days thanamong those at 49 days (3.3 versus 1.5%, P = 0.0193). The regimenappears as effective, in terms of high complete abortion rateand low continuing pregnancy rate, as any published alternative.This regimen has the benefit of being less costly as the doseof mifepristone is 67% lower and misoprostol is substantiallyless expensive than gemeprost. Additionally, misoprostol doesnot require special transport or storage requirements. As such,the combination of mifepristone and gemeprost.     

Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine

Comparative mutagenic and genotoxic effects of three antimalarialdrugs, chloroquine, primaquine and amodiaquine, were assessedin the Ames mutagenicity assay (in strains TA97a, TA100, TA102and TA104) and in vivo sister chromatid exchange (SCE) and chromosomeaberration (CA) assays in bone marrow cells of mice. These arethe most commonly used antimalarial drugs available at presentthroughout the world. The results of the bacterial mutagenicityassays showed a very weak mutagenic effect of all three drugsin Salmonella strains TA97a and TA100 both with and withoutS9 mix and in TA104 only with S9 mix. The results of the invivo SCE and CA assays indicate that these three drugs are genotoxicin bone marrow cells of mice. ³To whom correspodence should be addressed. Tel: +91 33 473 3491; Fax: +91 33 473 5197; Email: iichbio{at}giascl01.vsnl.net.in     

Effect of sodium arsenite on peripheral lymphocytes in vitro: individual susceptibility among a population exposed to arsenic through the drinking water

Arsenic (As) contamination in ground water has affected more than 19 countries. Approximately 36 million people in the Bengal delta alone are exposed to this toxicant via drinking water (>50 microg/l) and are at potential health risk. Chronic ingestion of As via drinking water is associated with occurrence of skin lesions, cancer and other arsenic-induced diseases in West Bengal, India. An in vitro cytogenetic study was performed utilizing chromosomal aberrations (CA) in lymphocytes treated with sodium arsenite (0-5 microM) in six symptomatic (having arsenic-related skin lesions) individuals, six age- and sex-matched As-exposed asymptomatic (no arsenic-related skin lesions) individuals and six control individuals with similar socio-economic status residing in non-affected districts of West Bengal with no evidence of As exposure. The mean As content in nails and hair was 9.61 and 5.23 microg/g in symptomatic, 3.48 and 2.17 microg/g in asymptomatic and 0.42 and 0.33 microg/g in the control individuals, respectively. The main aim of our study was to determine whether genotoxic effects differed in the lymphocytes of the control (no exposure to arsenic), asymptomatic and symptomatic individuals after in vitro treatment with sodium arsenite. Although both the exposed groups had chronic exposure to As through the drinking water, individuals with skin lesions accumulated more As in their nails and hair and excreted less in urine (127.80 versus 164.15 microg/l). The results show that sodium arsenite induced a significantly higher percentage of aberrant cells in the lymphocytes of control individuals than in the lymphocytes of both the exposed groups. Within the two exposed groups As induced higher incidences of CA in the symptomatic than the asymptomatic individuals. These results suggest that asymptomatic individuals have relatively lower sensitivity and susceptibility to induction of genetic damage by As compared with the symptomatic individuals.     

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het⁺ nif^?) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 mM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 mM. But at 1 mM, these amino acids were inhibitory. In addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine at 2 mM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N₂-fixing blue-green lagae may reveal the regulation of the heterocyst-nitrogenase sub-system.     

Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility

BACKGROUND: The aim of this study was to examine the role of apoptosis and reactive oxygen species (ROS) in inducing DNA damage in ejaculated spermatozoa. METHODS: We examined ejaculated spermatozoa from 31 patients examined for infertility and 19 healthy donors for apoptosis, production of ROS and DNA damage using annexin V binding, chemiluminescence assay and sperm chromatin structure assay. RESULTS: The percentage of spermatozoa that underwent apoptosis in the whole ejaculate and mature fraction was higher in the patients than in the donors (P<0.001 and P=0.009, respectively). Levels of ROS in the whole ejaculate and immature fraction were higher in the patients than in the donors (P=0.002 and P=0.009). Apoptosis was significantly correlated with ROS within patients in the whole ejaculate [r (95% confidence interval)=0.53 (0.19-0.86)] and in the mature [0.71 (0.39-1.00)] and immature spermatozoa [0.75 (0.45-1.00)]. Only apoptosis and the DNA fragmentation index (DFI) were significantly correlated within patients in the whole ejaculate [0.57 (0.18-0.97)]. CONCLUSIONS: DNA damage may be induced by oxidative assault. Apoptosis may not contribute significantly to the DNA damage.     

Pitfalls in the diagnosis of epithelioid sarcoma presenting in an unusual site: a case report

The cytologic appearance of epithelioid sarcoma in fine-needle aspiration biopsy cytology (FNABC) smears has not been extensively described. We report a case of epithelioid sarcoma in a 55-year-old male who presented with nodular swellings in the abdominal wall and scrotum. The scrotal swelling was subsequently demonstrated to arise from the vas deferens. We highlight the cytomorphologic, immunocytochemical, and histopathologic features of this rare lesion in an unusual site, with particular emphasis on the dilemma encountered in the cytodiagnosis of this lesion.     

Diagnostic value of routine semen analysis in clinical andrology

Infertility is a major health issue affecting over 48.5 million couples around the world, with the male factor accounting for about 50% of the cases. The conventional semen analysis recommended by the World Health Organization (WHO) is the cornerstone in the evaluation of male fertility status. It includes macroscopic and microscopic evaluation of the ejaculate, which reflects the production of spermatozoa in the testes, the patency of the duct system and the glandular secretory activity. Evaluation of seminal fructose, sperm vitality and leucocytes (Endtz test) are useful adjuncts to semen analysis that provide information on specific clinical conditions. Though several computer-assisted sperm analysis (CASA) systems have been developed, conventional methods for semen analysis are still widely accepted in clinical practice. This review summarises the conventional techniques used in routine semen analysis and their diagnostic value in clinical andrology.     

Protein profiling in unlocking the basis of varicocele-associated infertility

Varicocele is one of the major causes of male infertility and has a negative impact on spermatogenesis. The conventional semen analysis does not reveal the underlying subcellular mechanisms associated with defects in spermatozoa. Proteomics and bioinformatics analysis can be used to identify the molecular aetiologies associated with poor semen quality in varicocele patients. Mitochondrial dysfunction has been identified as the main factor affecting normal physiological functions of spermatozoa. This article discusses the proteomic studies of spermatozoa and seminal plasma in varicocele patients. Proteomics can identify potential spermatozoa and seminal plasma biomarkers in varicocele-mediated male infertility. In future, these protein biomarkers can be useful in the development of noninvasive diagnostic and therapeutic strategies for varicocele patients.