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Biomedical subjects

A Pawlowski

Publications and source records attributed to A Pawlowski.

At least 19 recordsLinked to original sources
PubMed2005

Nasal boost with adjuvanted heat-killed BCG or arabinomannan-protein conjugate improves primary BCG-induced protection in C57BL/6 mice.

Today it is generally accepted that the Bacillus Calmette-Guerin (BCG) vaccine protects against childhood tuberculosis (TB) but this immunity wanes with age, resulting in insufficient protection against adult pulmonary TB. Hence, one possible strategy to improve the protective efficacy of the BCG vaccine would be to boost in adulthood. In this study, using the mouse model, we evaluated the ability of two new TB vaccine candidates, heat-killed BCG (H-kBCG) and arabinomannan-tetanus toxoid conjugate (AM-TT), given intransally in a novel Eurocine adjuvant, to boost a primary BCG-induced immune response and to improve protection. Young C57BL/6 mice were vaccinated with conventional BCG and, 6 months later, boosted intranasally with adjuvanted H-kBCG or AM-TT, or subcutaneously with BCG. Ten weeks after the booster, mice were challenged intravenously with M. tuberculosis (Mtb) strain H37Rv. In spleens, there was a significant reduction of cfu counts in mice boosted with either H-kBCG or AM-TT vaccines compared to the non-boosted BCG-vaccinated mice. None of the boosting regimens significantly reduced bacterial loads in lungs, compared to non-boosted BCG vaccination. However, the extent of granulomatous inflammation was significantly reduced in the lungs of mice that received two of the booster vaccines (AM-TT and conventional BCG), as compared with sham-vaccinated mice. All boosted groups, except for mice boosted with the AM-TT vaccine, responded with a proliferation of spleen T cells and gamma interferon production comparable to that induced by a single BCG vaccination.

Administration, Intranasal↗
PubMed2005

Low dose chronic Schistosoma mansoni infection increases susceptibility to Mycobacterium bovis BCG infection in mice.

The incidence of mycobacterial diseases is high and the efficacy of Bacillus Calmette Guerin (BCG) is low in most areas of the world where chronic worm infections are common. However, if and how concurrent worm infections could affect immunity to mycobacterial infections has not been elucidated. In this study we investigated whether infection of mice with Schistosoma mansoni could affect the ability of the animals to control Mycobacterium bovis BCG infection and the immune response to mycobacterial antigens. BALB/c mice subclinically infected with S. mansoni were challenged with M. bovis BCG via the intravenous route. The ability of the animals to contain the replication of M. bovis BCG in their organs, lung pathology as well as the in vitro mycobacterial and worm antigen induced immune responses were evaluated. The results showed that S. mansoni coinfected mice had significantly higher levels of BCG bacilli in their organs and sustained greater lung pathology compared to Schistosoma uninfected controls. Moreover, Schistosoma infected mice show depressed mycobacterial antigen specific Th1 type responses. This is an indication that chronic worm infection could affect resistance/susceptibility to mycobacterial infections by impairing mycobacteria antigen specific Th1 type responses. This finding is potentially important in the control of TB in helminth endemic parts of the world.

Animals↗
PubMed2004

Immunization with heat-killed Mycobacterium bovis bacille Calmette-Guerin (BCG) in Eurocine L3 adjuvant protects against tuberculosis.

The current live attenuated vaccine against tuberculosis, BCG, poses a risk of disseminated infections in immunocompromised subjects. Therefore, in this study we compared the protective effect of a heat-killed bacille Calmette-Guerin (H-kBCG) vaccine given in a new adjuvant (Eurocine L3) with the protection provided by the conventional live attenuated BCG vaccine in mice (C57BL/6 and BALB/c) challenged with virulent Mycobacterium tuberculosis (strain Harlingen). The H-kBCG vaccine alone, in accordance with earlier studies, did not give any or only gave slight protection compared to sham-vaccinated controls. However, the same vaccine given with Eurocine L3 adjuvant, either formulated as a suspension or as an emulsion, afforded significant levels of protection. This protection was at least as good as that of the control live attenuated BCG vaccine. The Eurocine L3 adjuvant is approved for human use as a nasal vaccine adjuvant and a successful phase I trial with nasal immunization with diphtheria vaccine has recently been performed in Sweden. Here we show that, in mice, intranasal priming with H-kBCG in Eurocine L3 adjuvant followed by intranasal booster resulted in the same level of protection as subcutaneous priming followed by intranasal booster. All H-kBCG formulations in the Eurocine L3 adjuvant elicited mycobacterial antigen-specific serum IgG and IFN gamma responses. In general, among the different vaccine formulation(s) in the Eurocine L3 adjuvant those that produced a relatively high Th2 response, as measured by IgG1/IgG2a ratio and IFN gamma production in vitro, were the most protective. In conclusion, H-kBCG in Eurocine L3 adjuvant could represent a safe and a more stable alternative to the conventional live BCG vaccine.

Adjuvants, Immunologic↗
PubMed2004

A mycobacterial lipoarabinomannan specific monoclonal antibody and its F(ab') fragment prolong survival of mice infected with Mycobacterium tuberculosis.

Lipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis. High antibody titres against LAM are often seen in active tuberculosis (TB). The role of such LAM-specific antibodies in the immune response against TB is unknown. Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab')(2) fragment directed against LAM from M. tuberculosis strain H37Rv. MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M. tuberculosis strain Harlingen as well as to M. tuberculosis H37Rv. The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5.2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM. In passive protection experiments BALB/c mice were infected intravenously with M. tuberculosis Harlingen. MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria. The antibody proved to be protective against the M. tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival.

Animals↗
PubMed2001

Rapid diagnosis of tuberculosis by detection of mycobacterial lipoarabinomannan in urine.

There is an urgent need for improved tools for laboratory diagnosis of active tuberculosis (TB). Here, we describe two methods, a catch-up ELISA and a dipstick test based on the detection in urine of lipoarabinomannan (LAM). LAM is a major and specific glycolipid component of the outer mycobacterial cell wall. Preliminary experiments showed that LAM is excreted in the urine of mice injected intraperitoneally with a crude cell wall preparation of Mycobacterium tuberculosis. Both methods were highly sensitive, detecting LAM at concentrations of 1 ng/ml and 5 pg/ml, respectively. Of 15 patients with active TB, all showed intermediate to high levels of LAM in their urine (absorbance values from 0.3 to 1.2, mean 0.74). Only one sample showed an absorbance value below the chosen cut off value of 0.4. All but one of the urine samples from 26 healthy nursing workers exhibited OD value below 0.4 cut off. These methods may prove valuable for rapid and simple diagnosis of TB in particular in developing countries lacking biosafety level 3 (BSL3) facilities.

Agglutination Tests↗
PubMed2000

Preparation of pneumococcal capsular polysaccharide-protein conjugate vaccines utilizing new fragmentation and conjugation technologies.

There is a global urgent need for a new efficient and inexpensive vaccine to combat pneumococcal disease, which should also be affordable in developing countries. In view of this need a simple low-cost technique to prepare such a vaccine was developed. The preparation of serotype 14 and 23F pneumococcal capsular polysaccharide (PnPS)-protein conjugates to be included in a forthcoming multivalent PnPS conjugate vaccine is described. Commercial lots of PnPSs produced according to Good Manufacturing Practice from Streptococcus pneumoniae serotype 14 (PS14) and 23F (PS23F) were partially depolymerized by sonication or irradiation in an electron beam accelerator. The PnPS fragments were conjugated to tetanus toxoid (TT) using a recently developed conjugation chemistry. The application of these new simple, efficient and inexpensive fragmentation and conjugation technologies allowed the synthesis of several PnPS-protein conjugates containing PnPS fragments of preselected sizes and differing in the degree of substitution. The PS14TT and PS23FTT conjugate vaccine candidates were characterized chemically and their immunogenicity was evaluated in rabbits and mice. All PnPS conjugate vaccines, unlike the corresponding plain polysaccharides, produced high IgG titres in both animal species. The PS14TT conjugates tended to be more immunogenic than the PS23FTT conjugates. The immune response to the PS14TT conjugates, but not to the PS23FTT conjugates, was related to the size of the conjugated polysaccharide hapten. Both types of conjugates elicited strong booster effects upon secondary immunizations, resulting in high IgG1, IgG2a and IgG2b titres.

Acetylation↗
PubMed1999

Electron beam fragmentation of bacterial polysaccharides as a method of producing oligosaccharides for the preparation of conjugate vaccines.

End-group mediated conjugation of bacterial polysaccharides (PSs) to carrier proteins containing T-helper cell epitopes renders such polysaccharides immunogenic also in young infants. Optimal construction of such conjugate vaccines requires fragmentation of the PS prior to the coupling reaction. In the present study a general simple and inexpensive method for the fragmentation of PSs is presented. It is based on the irradiation of isolated PSs in an electron beam accelerator. Exposure of isolated pneumococcal capsular polysaccharides (PnPSs) to ionizing radiation resulted in their partial depolymerization in a radiation dose-dependent manner. Radiation, unlike sonication, generated PnPS fragments of molecular size lower than 50 kDa and as small as 1.5 kDa when high radiation doses were used. These PnPS fragments have terminal reducing groups that can be easily used for chemical activation and subsequent coupling to any chosen carrier protein. The radiation-produced PnPS fragments retained their antigenic epitopes, when compared to native, full-size PnPSs as determined by enzyme-linked immunoassay.

Animals↗
PubMed1999

A new method of non-cross-linking conjugation of polysaccharides to proteins via thioether bonds for the preparation of saccharide-protein conjugate vaccines.

Bacterial polysaccharides, including capsular polysaccharides, are poor immunogens particularly in young infants. However, conjugation of bacterial polysaccharides to immunogenic carrier proteins generally results in conjugates that induce strong antipolysaccharide T-helper-cell dependent immune responses, also in young infants. The magnitude of the response and the extent of the T-helper-cell dependency is related to the chemical characteristics of the particular conjugate such as presence or absence of polysaccharide-protein cross-linking, presence or absence of spacer arms, character of spacer arms, type of carrier protein, size of conjugated polysaccharide hapten and molar degree of substitution. In the present study a new, general and simple method for the preparation of poly- and oligosaccharide-protein conjugates is presented. This new method is based on spacer-introducing chemistry that allowed for conjugation of a model polysaccharide, dextran, ranging in size from 0.5 to 150 kDa, to tetanus toxoid (TTd). The developed conjugation method involves derivatization of polysaccharide with 2-iminothiolane (2-IT) and activation of carrier protein, such as TTd, with N-hydroxysuccinimide ester of bromoacetic acid. Reaction rates and accordingly the substitution of the conjugates, could be controlled by varying time, pH and concentration of the reactants. Unlike direct reductive amination, the 2-IT based conjugation technology is fast and made it possible to couple fairly large polysaccharides to TTd.

Dextrans↗
PubMed1991

Effects of UV radiation on the ultrastructure of human common pigmented naevi and lentigines.

In order to investigate how sunlight may affect naevi and lentigines, their melanocytes and the basement membrane, three irradiation protocols were applied directly to ten naevi and five lentigines on 2 subjects. Neither volunteer had sufficient naevi and lentigines to be able to use the three irradiation protocols on each of the subjects. Skin biopsies were fixed in glutaraldehyde followed by osmium tetroxide, thin-sectioned and examined in a Hitachi H-7000 transmission electron microscope. Following 14 consecutive single exposures of 3 MED of UVB or single exposures followed by 25 J/cm2 of UVA, 350 J/cm2 UVA with either 2040 or 2280 mJ/cm2 UVB, the basement membrane maintained its continuity. Melanocytes were not observed on the dermal side of the epidermal-dermal junction. UVA irradiation stimulated reinforcement of the basement membrane zone by collagen fibers. Centrioles found in melanocytes following irradiation suggest that these melanocytes maybe undergoing mitosis. Dermal fibroblasts were found to contain comparatively large quantities of melanin pigment. The pigment contained in these fibroblasts may in fact constitute an additional barrier against UV irradiation.

Adult↗
PubMed1989

Effects of IGF-I, rGH, FGF, EGF and NCS on DNA-synthesis, cell proliferation and morphology of chondrocytes isolated from rat rib growth cartilage.

The effects of IGF-I, rGH, FGF, EGF and NCS on DNA-synthesis were analyzed in resting, proliferative and hypertrophic chondrocytes obtained by fractionation. Proliferation and morphology were studied on non-fractionated cells. The highest stimulation of DNA-synthesis was induced by NCS followed by IGF-I at all stages of chondrocyte differentiation. DNA-synthesis was also stimulated by a low concentration of FGF (1 microgram/1) in proliferative and hypertrophic chondrocytes, while FGF in a higher concentration (10 micrograms/1) had no significant mitogenic effect. Cell proliferation was stimulated by both NCS and IGF-I, whereas FGF and EGF only caused morphological changes. Our data indicate that IGF-I is the main serum growth factor regulating growth and proliferation by interacting with chondrocytes at all stages of differentiation.

Animals↗
PubMed1988

Effects of IGF-I, EGF, and FGF on proteoglycans synthesized by fractionated chondrocytes of rat rib growth plate.

The effects of insulin-like growth factor (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF), or 10% newborn calf serum (NCS) on the amount and structure of the proteoglycans synthesized by fractionated chondrocytes from rat growth plate were investigated. Proliferative cells (fraction II) or resting cells (fraction III) synthesized more proteoglycans than hypertrophic cells (fraction I). Addition of IGF-I to the cultures increased proteoglycan synthesis more than addition of EGF or FGF. EGF and FGF induced synthesis of proteoglycans of smaller molecular size with a lower proportion of aggregates. The size of the constituent glycosaminoglycan chains did not differ between control and growth factor-treated cells. The present study demonstrates that proteoglycan structure and synthesis are modified by growth factors to different extents, depending on the maturation stage of the target cells.

Animals↗
PubMed1988

Ultrastructural changes produced in Ehrlich ascites carcinoma cells by ultraviolet-visible radiation in the presence of melanins.

Irradiation of Ehrlich ascites carcinoma (EAC) cells in the presence of pheomelanin, i.e., red hair melanin (RHM), has been reported to produce extensive cell lysis. Irradiation in the presence of eumelanin, i.e., black hair melanin (BHM), or irradiation in the absence of either type of melanin did not produce this effect. We observed that RHM particles penetrated the cell membrane without apparent structural damage to the cell or the cell membrane. Irradiation of the cells in the absence of melanin did not produce any changes in the ultrastructure of the cells. Incubation of the cells in the dark in the presence of RHM produced only minor structural, mainly cytoplasmic changes. Irradiation of the cells in the presence of RHM produced extensive ultrastructural changes prior to complete cell lysis; these changes were more severe than the effects of incubation of the cells in the dark in the presence of RHM. When the cells incubated in the dark or irradiated in the presence of latex particles or either one of the eumelanins particles, viz. BHM or synthetic dopa melanin, these particles did not penetrate into the cells or produce any ultrastructural changes. These particles were in fact not even ingested by the cells.

Animals↗
PubMed1987

Human tattoo. Electron microscopic assessment of epidermis, epidermal-dermal junction, and dermis.

Ultrathin serial sections of human biopsy specimens, taken at 24 hours, 1 month, and 1, 3, and 40 years post-tattooing were examined under the electron microscope. The ink particles found in cells were measured and compared with control ink particles embedded in agar. Freshly tattooed skin showed an inflammatory reaction followed by ultrastructural necrosis. The time of healing, about 1 month from introduction of ink to complete healing, has been divided into three phases: inflammatory reaction and necrosis, formation of basement membrane, and normal epidermis and dermis. Once the skin showed normal ultrastructure, ink particles were found only in dermal fibroblasts.

Basement Membrane↗
PubMed1987

Cell fractions from rat rib growth cartilage. Morphological and X-ray microanalytical investigation.

For in vitro studies of differentiation and proliferation of chondrocytes, a need arose to prepare enriched cell fractions for analysis of the effects of different growth promoting factors. Therefore chondrocytes were isolated from rat rib growth cartilage in young rats by collagenase digestion and subsequently three cell fractions were prepared by centrifugation in a step gradient of Percoll. In a previous paper, matrix molecules synthesized by each fraction were characterized biochemically. In the present study, ultrastructure and elemental content of the fractionated chondrocytes were analyzed by scanning electron microscopy, transmission electron microscopy, and energy dispersive X-ray microanalysis. DNA-synthetic activity was measured by means of autoradiography of 3H-thymidine incorporating cells. Both morphology and elemental concentrations differed between fractions and were characteristic for each fraction. The cell fraction with the lowest density consisted of large, polygonal cells that became flattened in culture. The reduced synthetic activity and markedly lowered K content of these cells suggest that they originate from the hypertrophic zone of the growth plate. The cells in the fraction with the highest density were rather homogeneous in size and shape, and had a well developed rough endoplasmic reticulum and Golgi complex, characteristic of proliferating and resting chondrocytes. Concentrations of P and K were also significantly higher in this fraction. The fraction with intermediate density contained an admixture of cells with a predominance of proliferating cells with high DNA-synthetic activity.

Animals↗
PubMed1986

Cell fractions from rat rib growth cartilage. Biochemical characterization of matrix molecules.

In an attempt to isolate and characterize the putative target cells for growth hormone, chondrocytes were isolated from rat rib growth cartilage and fractionated by centrifugation in a discontinuous Percoll gradient. This resulted in three cell fractions with differing properties. The fraction with the lowest density consisted mainly of large, lipid-containing cells which became flattened in subsequent culture. The cells in this fraction were fair collagen producers but synthesized only minor amounts of proteoglycans and apparently no proteoglycan aggregates. These cells probably originate in the hypertrophy zone of the growth plate. The fraction with highest density, on the other hand, consisted of small cells which upon cell culture became polygonal and surrounded with refractile matrix. They synthesized less collagen, but more proteoglycans than the low-density fraction. The proportion of proteoglycan aggregates was rather low (10-20%) but otherwise the proteoglycans were similar to those synthesized by other chondrocytes. This cell fraction was tentatively identified as cells from the upper part of the growth plate. Finally, the middle fraction was heterogeneous, consisting of cells of varying shape. This fraction synthesized large amounts of both collagen and proteoglycans. In all three cell fractions, type II collagen predominated. There were also minor amounts of (1a, 2a, 3a) collagen, and, in the two denser fractions, of type I collagen.

Animals↗
PubMed1986

Human melanocytic naevi. II. Depth of dermal protrusion: comparative measurements of the basement membrane zone and diameters of intracellular microfibrils using a microcomputer analysis system.

Thirteen acquired melanocytic naevi and five control skin biopsies were taken and fixed for observation both in the light and electron microscope. The thickness of the basement membrane, the distance of the naevus protrusion into the dermis, the number of anchoring fibrils attached to the lamina densa and the diameter of intracellular filaments in the naevus cells were measured. Comparative measurements were made in the electron microscope, from micrograph negatives and from micrographs. A non dedicated microcomputer digital analysis system developed specifically from this type of analysis, was also used.

Basement Membrane↗