Bulgarian Bone Marrow Donors Registry—past and future directions

Recently Bulgarian Bone Marrow Donors Registry (BBMDR) has been established and since August 2005 it has been a member of Bone Marrow Donors Worldwide. Currently the number of healthy donors included in the BBMDR is relatively low. All donors included in the BBMDR are typed for HLA-A, -B, -DRB loci. Phylogenetic analysis based on HLA allele frequencies shows that Bulgarians were characterized with closest genetic similarity to Macedonians, Greeks, Romanians, Cretans and Sardinians in comparison to the other European and Mediterranean populations. On the contrary the second largest ethnic minority–the Roma were the closest to the other Roma populations and North Indians. These differences were due to the predominance of alleles and haplotypes that are specific for the Asian and the other Roma populations. These specific genetic profiles in the Bulgarian ethnic minorities justify the need of an adequate representation of minorities in BBMDR. Future directions for BBMDR development are discussed, including an increase of the total number of donors and these for ethnic minorities, as well the enhancement of the level of resolution of the HLA typing for the donors in the registry.     

Survival of Escherichia coli under lethal heat stress by L-form conversion

Transition of bacteria to cell wall deficient L-forms in response to stress factors has been assumed as a potential mechanism for survival of microbes under unfavorable conditions. In this article, we provide evidence of paradoxal survival through L-form conversion of E. coli high cell density population after lethal treatments (boiling or autoclaving). Light and transmission electron microscopy demonstrated conversion from classical rod to polymorphic L-form shape morphology and atypical growths of E. coli. Microcrystal formations observed at this stage were interpreted as being closely linked to the processes of L-form conversion and probably involved in the general phenomenon of protection against lethal environment. Identity of the morphologically modified L-forms as E. coli was verified by species specific DNA-based test. Our study might contribute to a better understanding of the L-form phenomenon and its importance for bacterial survival, as well as provoke reexamination of the traditional view of killing strategies against bacteria.     

Investigations of mannan and glucan in the cell wall ofCandida boidinii cultivated on methanol

The polysaccharide components (mannan and glucan) in the cell wall ofCandida boidinii M 363 grown on methanol and glucose as control were investigated using electron microscopy, cytochemical and biochemical methods. An ultrastructural rearrangement of the polymers in the cell wall of yeasts cultivated on methanol in comparison to those cultivated on glucose was established. The morphological changes correlate to the quantitative changes in the polysaccharide constituents of the cell wall. The forming and the role of thiosemihydrocarbazide (TSHC) — negative zones in theCandida boidinii cell wall cultivated on methanol media are discussed.     

Effect of the pesticides phosalone and lindane on the activity of some dipeptidases and disaccharidases in rat intestinal mucosa.

The intestinal dipeptidase and disaccharidase activities were investigated in 120 male albino rats of the Wistar strain after administration of 21 mumol.kg-1 body weight phosalone, 14.8 mumol.kg-1 body weight lindane and 10.5 mumol.kg-1 body weight phosalone combined with 7.4 mumol.kg-1 body weight lindane. The dipeptidase activity under the effect of these comparatively low doses of pesticides reveals slightly to moderate changes. The activity of intestinal disaccharidases after a 90-day phosalone and lindane treatment is markedly decreased, particularly that of sucrase. The mechanism of these changes remains unknown.     

Cell-wall-deficient forms ofStaphylococcus aureus as lung pathogens: an ultrastructural study

The interaction between stable protoplast L forms ofStaphylococcus aureus and the alveolar wall of infected rats was observed in the course of experimental pulmonary infection (days 3, 7, 14 and 30 p.i.). The L forms were successfully cultivated from bronchoalveolar lavage samples taken throughout the tested interval. The ultrastructural results demonstrated the ability of the L forms to invade the alveolar wall where they established, grew and reproduced mainly in the interstitium. The infection caused lung lesions: granulomas, focal fibroses and destruction of type I alveolar epithelial cells.     

Effects of the chromatographic fractions of the pig placental trophoblast on graft-versus-host reaction

The trophoblast has a significant role in regulation of immune reactions at the materno-fetal interface by producing biologically active substances. In our previous studies five fractions with immunomodulatory activities were isolated by gel chromatography from trophoblast of pig placentas. To confirm the immunomodulatory effect of these trophoblast fractions on allogeneic in vivo systems and to obtain more evidence for the relevance of their activity on the maternofetal interface, their effect was studied on graft-versus-host reaction (GVHR). To assess the GVHR, the primary and secondary popliteal lymph nodes assay was used in mice. In the primary GVHR, 100 microg protein of Fraction 2-5, mixed with 5 x 10(6) allogeneic spleen cells (C57BL/6), were injected into one of the foot pads of recipient (BALB/c) mice. The secondary GVHR was induced in F1 (BALB/c x C57BL/6) mice by injection of spleen cells of BALB/c mice intraperitoneally preimmunized with allogeneic cells. The GVHR was measured by the weight of lymph nodes and by the lymphocyte proliferation. Flow cytometric analyses of the cells in the nodes with GVHR and under the influence of Fraction 4 or 5 were performed using monoclonal antibodies. In the primary GVHR, Fraction 4 or 5, injected simultaneously with allogeneic spleen cells, significantly suppressed the lymph nodes reactivity. Fractions 4 and 5 inhibited the ability of the spleen cells of mice intraperitoneally preimmunized with allogeneic cells to induce secondary GVHR in F1 mice. The Fraction 2 and 3 had no effect on GVHR. The results revealed that a group of proteins with Mr 37-7 kDa, isolated from trophoblast of pig placenta, strongly suppressed popliteal lymph node reactivity in the primary and secondary GVHR. The data provide convincing evidence for these fractions in vivo activity, for their effect across the species barrier and suggest the relevance of the same reactions on the materno-fetal interface.     

Mutant Hansenula polymorpha strain with constitutive alcohol oxidase and peroxisome biosynthesis

A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains--DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol x min(-1) x mg(-1) protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.     

Persistence of Staphylococcus aureus L-form during experimental lung infection in rats

The course of pulmonary infection in rats infected by intranasal inoculation with a Staphylococcus aureus stable protoplast L-form was studied. Blood and bronchoalveolar samples were taken on days 3, 7, 14 and 30 after challenge and were investigated by microbiological, electron-microscopic, cytochemical and cytometric methods. The electron microscopic data and isolation of L-form cultures from bronchoalveolar samples at all experimental times demonstrated the ability of S. aureus L-form cells to internalize, replicate and persist in the lungs of infected rats to the end of the observation period, in contrast to the S. aureus parental form. It was found that persisting L-form evoked ineffectual phagocytose by alveolar macrophages and low but long-lasting inflammatory reaction in rats. The experimental model of pulmonary infection with S. aureus L-form suggests that the cell-wall-deficient bacterial forms may be involved in the pathogenesis of chronic and latent lung infections.     

Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress

Bacteria can, under certain conditions, enter into a cell-less state known as L-form conversion. This phenomenon is universal, but also recognized with difficultly by microbiologists. The current study addresses several aspects concerning the ability of tubercle bacilli to use L-form conversion as a unique adaptive strategy to survive and reproduce under unfavorable conditions. Nutrient starvation of M. tuberculosis in vitro followed by passages in Middlebrook 7H9 semisolid medium was used for stress induction and the selective isolation of mycobacterial L-form variants. Light and electron microscopy images evidence the peculiar characteristics of mycobacterial L-forms. For example, mycobacterial L-forms were observed to lose their acid-fastness and change their morphology. In addition, wide morphological variability, the presence of large and elementary bodies, coccoids and small granular forms, as well as the appearance of unusual modes of irregular cell division were observed. Unlike classical tubercle bacilli, L-form variants grew and developed typical "fried-egg" colonies faster. L-forms were verified as M. tuberculosis by spoligotyping. The results provide insights into the nature of L-form phenomena in M. tuberculosis and link them to the mechanisms allowing mycobacterial survival under stress.