From testicular biopsy to human embryo.

The aim of the study was to investigate the role of a testicular biopsy in the diagnosis and therapy of infertile men with a non-obstructive azoospermia. Overall, 70 testicular biopsies from infertile men were analysed. Samples were obtained by the "open testicular biopsy" method. After dissection, several pieces of the tissue were immediately immersed into the Sperm Prep Medium (Medi-Cult) and fixative (5.5% buffered glutaraldehyde). Tissue samples transported in Sperm Prep Medium were plunged into Sperm Freezing Medium (Medi-Cult) and were stored in liquid nitrogen for potential in vitro fertilization procedures. The tissue was also processed for semithin sections and transmission electron microscopy. Semithin sections from 8 infertile patients demonstrated regular testis structure and fully preserved spermatogenesis (control biopsies). In the remaining 62 cases, spermatogenesis was impaired and a variety of pathological changes could be seen: disorganization and desquamation of spermatogenic cells, spermatid or spermatocyte "stop", spermatogonia only, "Sertoli cells only" or tubular fibrosis. However, in 65% of cases (despite the above mentioned changes of seminiferous epithelium) foci of preserved spermatogenesis could be detected. These cases were classified as "mixed atrophy" of seminiferous tubules. In 63% of infertile patients, a successful extraction of sperm from the biopsy could be performed. In azoospermic patients, histological analysis of testicular biopsy proved to be very useful in terms of diagnosis as well as therapy, i.e. for further in vitro fertilization procedures.

Effects of various cryopreservation media and freezing-thawing on the morphology of rat testicular biopsies.

Currently, testicular sperm extraction is successfully combined with intracytoplasmic sperm injection into the oocyte (ICSI). Several pieces of a testicular biopsy can be frozen and thawed until the ICSI attempt. In this study, the effects of freezing-thawing on the morphology of rat testicular biopsies stored in different cryopreservation media were analysed. Each cryopreservation medium contained glycerol and/or dimethyl sulfoxide (DMSO) as cryoprotectants. In general, both glycerol and DMSO, when applied at moderate concentrations (6-25%), preserved the structure of the seminiferous epithelium. The freezing-thawing procedure had no significant effect on tubular diameter; however, it caused a 'folding' of the lamina propria and notable damage to Sertoli cells, spermatogonia and spermatocytes. Round and elongated spermatids and spermatozoa displayed occasional nuclear damage, vacuolization, and shrinkage/swelling of the cytoplasm. However, the vast majority of these cells maintained their normal structure in nearly all the applied cryomedia. It is concluded that freezing-thawing of testicular biopsies, and the cryopreservation medium, have a significant impact on the structure of the seminiferous epithelium, particularly on its basal compartment.

Cell density-dependent changes of glycosphingolipid biosynthesis in cultured human skin fibroblasts.

In this study, the glycosphingolipid biosynthesis was investigated in the sparse and the confluent cell populations of cultured human skin fibroblasts. The human skin fibroblast cell populations were metabolically pulse labeled with (14)C-galactose (48 h). The amounts of (14)C-radioactivity (cpm) incorporated into extracted and purified total cellular glycosphingolipid fractions were counted by beta-scintillation and the individual glycosphingolipid species were separated by high performance thin layer chromatography and visualized by autoradiography. The relative labeling (%) of individual newly synthesized glycosphingolipid species was detected by densitometric scanning of autoradiographic glycosphingolipid patterns. The incorporation of (14)C-label into total glycosphingolipids per cell increased significantly as the cell-density increased, referring to five fold higher rate of glycosphingolipid biosynthesis de novo in cells at confluency vs. sparse populations. The total newly synthesized glycosphingolipid pattern (100%) of sparse cell populations showed a significant predominance of the gangliosides (70%) over the neutral glycosphingolipids (30%), with ganglioside GM2 as the major species followed by monohexosyl-ceramide. Oppositely, the newly synthesized neutral glycosphingolipids (67%) predominated over the gangliosides (33%) in cells at confluency (contact inhibition). Cells reaching confluency were characterized by: (a) a dramatic increase of absolute amount of all newly synthesized neutral glycosphingolipid species, particularly the most abundant monohexosyl-ceramide and trihexosyl-ceramide, but also of the ganglioside GM3; (b) a drastic decrease of absolute amount of newly synthesized ganglioside GM2. The specific shift in newly synthesized glycosphingolipid pattern in cells reaching confluency suggests a down-regulation of biosynthetic pathway primarily at the level of N-acetylgalactosaminyl-transferase. A possible involvement of glycosphingolipids in cell density-dependent regulation of cell growth through establishment of the direct intermolecular intermembrane interactions is discussed.

[Antibiotic resistance of gram-negative bacteria isolated at a hospital intensive care unit]. / Kórházi intenzív osztályon izolált gram-negatív kórokozók antibiotikum rezisztenciájának alakulása.

The antibiotic sensitivity of Gram-negative facultatively pathogenic bacteria isolated between 1982-1986 in the respiratory intensive care unit of the 3rd department of pediatry of the "László" Hospital (Budapest) has been studied. Pseudomonas aeruginosa, Klebsiella, Acinetobacter calcoaceticus and Acinetobacter lwoffii were most frequently isolated both from the clinical samples (total 56.9%) and in samples obtained from hospital-hygienic check-examinations. 70-100% of the individual species was resistant to antibiotics used in Hungary. The gentamicin and tobramycin resistance of P. aeruginosa and A. lwoffii showed significant rate of increase. Favourable in vitro results were obtained only with ceftriaxon, ceftazidime, netilmicin and amikacin.

Origin and spread of Pseudomonas aeruginosa, Proteus and Klebsiella during twenty years in an infectious hospital.

Bacteriological examinations carried out in an infectious hospital revealed that the occurrence of Pseudomonas and Proteus grew 4-fold, and the rate of Klebsiella positive cultures 3.5-fold between 1958 and 1977. On the other hand, the occurrence of Staphylococcus aureus decreased to the half since 1961. The occurrence of Gram-negative facultative pathogens started to increase in the surgical wards in the fifties and the rise lasted until the mid-sixties. Pseudomonas aeruginosa was the most frequent among them. In contrast, Escherichia coli dominated and P. aeruginosa was the least frequent in the non-surgical wards. Here the Gram-negative facultative pathogens showed a more rapid increase and the incidence of P. aeruginosa and Proteus kept rising throughout the whole examination period. When Gram-negative facultative pathogens of hospital origin were colonizing, the proportion of sick persons versus symptomless carriers was significantly higher than in the case of extrahospital colonization on the basis of the records of 300 P. aeruginosa, 300 Proteus and 300 Klebsiella positive patients. This proportion changed parallel with the rate of the strains of hospital origin. The number of patients who acquired P. aeruginosa, Proteus or Klebsiella extrahospitally, kept continuously rising between 1958 and 1971. Thus, the advance of Gram-negative facultative pathogens is due not ony to nosocomial causes.

Extrahospital and intrahospital factors predisposing to the spread and colonization in patients of Pseudomonas aeruginosa, Proteus and Klebsiella in an infectious hospital.

The number of patients admitted to hospital who harbour Pseudomonas aeruginosa, Proteus and Klebsiella, keeps rising. Of the factors predisposing to colonization, only diabetes and antibiotic therapy exert their effect equally in extrahospital and intrahospital environment. Malignant diseases, immune suppressive therapy and instrumental interventions play a predominant role in the hospital. In extrahospital environment, infancy and old age, poor general condition as well as in almost half of the cases, an inflammatory process caused by viruses or bacteria was found to create favourable conditions for the colonization of facultative pathogens. One of the main sources of the Gram-negative facultative pathogens studied was the faeces of enteric patients in the hospital. The frequency of P. aeruginosa, Klebsiella and Proteus positive cultures rose parallel in the faecal and non-faecal bacteriological samples in the period 1958 to 1977. The seasonal changes observed in the frequency of positive cultures revealed that the Gram-negative facultative pathogens had increased in number first in the enteral wards, spreading subsequently to the medical and paediatric wards, and finally they appeared in a high number in the surgical wards, originating from patients transferred there from the medical or paediatric wards.

Leukocyte migration inhibition by a specific antigen in human typhoid fever.

Cellular reactivity to heat-killed Salmonella typhi antigen was investigated by the leukocyte migration inhibition (LMI) method in 33 S. typhi infected patients and in 32 control persons. In the typhoid group a statistically significant LMI was observed as compared to the members of the control group. A correlation was found between the level of the cellular sensitivity and the time elapsed between onset of the disease and performance of the test. Previous typhoid vaccination had no influence on the LMI. No correlation was found between the agglutinin titres and the sensitivity demonstrated by the LMI test. The value of the method in studies of cellular immunity in typhoid fever is discussed.

Post-therapeutic levels of antimicrobial drugs in faeces.

After oral administration for 3 days, furazolidone disappeared from the faeces on the first, polymyxin B and ampicillin on the fourth, and trimethoprim + sulphamethoxazole and nalidixic acid on the fifth day.