Search PubMed⌕ Search

Biomedical subjects

K Holmberg

Publications and source records attributed to K Holmberg.

At least 55 records · Page 3Linked to original sources

Regulation of expression of galanin and galanin receptors in dorsal root ganglia and spinal cord after axotomy and inflammation.

Galanin can normally be detected only in a few dorsal root ganglion (DRG) neurons, but it is dramatically upregulated after peripheral nerve injury in both rat and monkey. Galanin is stored in large dense core vesicles, which after axotomy are often found close to the membrane of afferent nerve endings in the dorsal horn. In the monkey there is an increase in galanin in many nerve terminals in the superficial dorsal horn after axotomy, but such an increase is more difficult to detect in the rat. Galanin is also present in local dorsal horn neurons, where it is upregulated by peripheral inflammation. Both galanin-R1 and galanin-R2 receptor mRNAs are expressed in rat DRGs, mainly in, respectively, large and small DRG neurons. Galanin-R1 receptor mRNA is downregulated in DRG neurons after axotomy, and a small decrease in galanin-R2 receptor mRNA levels can also be seen. After peripheral tissue inflammation galanin-R1 receptor mRNA levels decrease and galanin-R2 receptor mRNA levels increase. The present results show that galanin and galanin receptors are present in sensory and local dorsal horn neurons and are regulated by nerve injury and inflammation. Galanin may therefore be involved in processing of pain information, primarily exerting analgesic effects. Whereas local dorsal horn neurons represent a defense system against inflammatory pain, we have proposed that a second defense system, against neuropathic pain, is intrinsic to DRG neurons.

Animals↗

Radiation induced chromosomal instability in human T-lymphocytes.

Chromosomal instability in proliferating mammalian cells is characterized by a persistent increase of chromosomal aberrations and rearrangements occurring de novo during successive cell generations. Recent results from many laboratories using a variety of cells and cytogenetic end points show that this phenotype can be induced by low as well as high LET irradiation. A typical feature of chromosomal instability in primary human G0-lymphocytes exposed to gamma-irradiation at both high dose rate (45 Gy h-1) and low dose rate (0.024 Gy h-1) is the appearance of novel aberrations in the clonal progeny of the irradiated cell, many generations after the exposure. The same phenotype was observed in lymphocytes that were allowed to recover for 5 days in G0 after the radiation exposure, as well as in hprt-mutant T cell clones. These results demonstrate that neither the acute genotoxic stress caused by high dose rate as compared to low dose rate irradiation, nor a hypothesized conflict between mitogen induced growth stimulation and growth arrest due to radiation damage, seem to be critical conditions for the development chromosomal instability in these cells. In contrast to observations in other cells, no evidence of a persistent decrease of cloning ability was observed in the progeny of radiation-exposed human lymphocytes, and no alteration was observed in their sensitivity to a second radiation exposure. Furthermore, the frequency of CA-repeat length variation at three loci was not increased in the progeny of X-irradiated T cells as compared to non-irradiated cells, which indicates that microsatellite instability is not part of the chromosomal instability phenotype in human T-lymphocytes.

Chromosome Aberrations↗

Coexistence of enkephalin- and tyrosine hydroxylase-like immunoreactivities in nerve fibers of the temporomandibular joint capsule of the guinea pig.

BACKGROUND: The innervation of joints has been suggested to play an important role for their normal function as well as for the pathogenesis of chronic arthritic conditions. It is therefore important to elucidate the functional anatomy of this innervation. METHODS: The expression of methionine enkephalin-like immunoreactivity (ENK-LI) and tyrosine hydroxylase (TH)-LI as well as their possible colocalization were examined in the temporomandibular joint of the guinea pig: RESULTS: Nerve fibers with ENK-LI were demonstrated in the synovium of the guinea pig temporomandibular joint capsule with the use of indirect immunofluorescence. The ENK+ fibers were found close to the surface of the synovial membrane, deeper in the synovium, and at the walls of blood vessels. Coexistence of ENK- and TH-LI was observed frequently in the synovial nerve fibers. After removal of the superior cervical ganglion (SCG), the ENK-containing nerve fibers were reduced substantially in number on the operated side or were completely absent if the cervical sympathetic trunk was also excised. CONCLUSIONS: The findings indicate that the majority of fibers with ENK-LI derive from the SCG ENK may act as a neuromodulator on the catecholaminergic nerves and may also have an analgesic effect in the joint.

Animals↗

The innervation of the synovium of the knee joint in the guinea pig: an immunohistochemical and ultrastructural study.

The innervation of the knee joint synovial membrane of the guinea pig, i.e., the synoviocyte layer, the subjacent connective tissue and the connective tissue region beneath, was analyzed with immunohistofluorescence and electron microscopy. A screening of the innervation with antibodies against the general axon marker -- protein gene product (PGP) 9,5 -- revealed the presence of nerve fibers distributed in various regions of the knee joint synovial membrane. Confirming previous studies, some of these nerve fibers stained with antibodies to tyrosine hydroxylase (TH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP). In addition, dynorphin (DYN)-containing fibers were detected, which have not been reported previously in normal joints. In general, the immunoreactive fibers were observed close to the synoviocytes and at blood vessels. Fibers with colocalization of NPY- and TH-like immunoreactivities (LIs), as well as of DYN- and TH-LIs were demonstrated. In the electron microscope, bundles of unmyelinated fibers as well as single fibers were found in the connective tissue region below the synoviocytes. Varicose parts of the nerve fibers contained mainly small, clear vesicles. Small and large dense-cored vesicles were also seen, but less frequently. Denser portions of the plasma membranes of some axons were observed in these regions, facing the extracellular space. Myelinated fibers were also observed in some nerve bundles. These findings emphasize the complex innervation of the synovial membrane, with nerve fibers containing a host of neuroactive substances. Altogether, these fibers are probably involved in many functions such as vasoregulation and control of synovial secretion in addition to being a source of mediators in joint inflammation.

Animals↗

The distribution of cGMP in the adrenal gland in the rat, guinea pig and mouse after stimulation with sodium nitroprusside.

Nitric oxide (NO) acts as an intercellular messenger molecule in the nervous system. In the adrenal gland sympathetic preganglionic fibers innervating the medulla, as well as intrinsic neural ganglion cells, contain nitric oxide synthase (NOS). Nitric oxide stimulates the soluble enzyme guanylate cyclase forming cyclic GMP (cGMP). Using sodium nitroprusside (SNP) as nitric oxide donor we have studied the putative target cells for nitric oxide in the rat adrenal gland, both in vivo and in vitro. The guinea pig and a few mouse adrenal glands were studied after SNP perfusion for comparison. Our results show that after vascular perfusion with a high concentration (3 mM) of SNP both noradrenaline and adrenaline chromaffin cells express cGMP-like immunoreactivity in all three species. After incubation of rat adrenal slices with SNP primarily the noradrenaline chromaffin cells are cGMP-positive. In contrast, detectable levels of cGMP-like immunoreactivity were not found in neuronal ganglion cells. In the adrenal cortex cGMP-like immunoreactivity was seen in blood vessel walls, in small cells with processes forming a reticular network, at least partly presumably representing endothelial cells, as well as in some presumable nerve terminals. These findings support the view that chromaffin cells, especially the noradrenergic ones and blood vessels, are targets for nitric oxide in the adrenal gland.

Adrenal Glands↗

Surface characterization of biomedical materials by measurement of electroosmosis.

This paper reviews recent studies by the authors on the surface characterization of biomedically significant materials through electroosmosis determination. The surfaces studied include transparent and nontransparent materials such as quartz, ceramics, paper, and cast polymer capillaries, slides, and particles, in both native and surface modified form. The method is nondestructive, relatively fast, mechanistically simple, automatable to varying degrees, and can be used to analyze samples under physiologically compatible conditions. New experimental and mathematical modeling approaches allow estimates to be obtained with regard to the surface density and pK of various chemical groups, as well as the thickness of polymer or other surface coatings. Surface modifications which may be characterized include, covalent alteration via radiofrequency plasma discharge or organosilane grafting, noncovalent alteration via polymer adsorption, and covalent grafting of neutral polymers, such as poly(ethylene glycol) or dextran. Results complement those from other surface analysis techniques, and correlate with physiologically significant phenomena such as protein adsorption.

Biocompatible Materials↗

Chromosomal instability in in vivo radiation exposed subjects.

PURPOSE: To investigate whether delayed chromosomal instability arises in human peripheral T lymphocytes exposed in vivo to gamma-irradiation. MATERIALS AND METHODS: Long-term cultures were established from lymphocytes obtained from subjects involved in the radiological accident in Estonia in 1994. Two individuals exposed to a high dose, one individual with low exposure and one apparently unexposed person were studied. Two Estonian controls not involved with the accident were also analysed. Cells were grown for 6-42 days and chromosomal aberrations were assessed from G-banded metaphases. In addition, FISH chromosome painting analysis was performed on short-term cultures established from whole blood. RESULTS: No obvious sign of chromosomal instability was observed in the in vivo follow-up of the frequencies of chromosomal aberrations in lymphocytes of radiation accident victims performed by the FISH technique (48 h cultures established at different time intervals after the exposure). However, when the lymphocytes were cultured long term in vitro, chromosomal instability was observed. There was no dose-response, and the appearance of chromosomally unstable cells in long-term cultures was also observed in a subject exposed to a dose of less than 0.1 Gy. Moreover, in contrast with previous findings, chromosomal instability was also observed in cells from non-exposed control individuals. The chromosomal changes observed in the controls were less complex than the aberrations in the cultures derived from individuals exposed to high doses. CONCLUSIONS: Chromosomal instability was observed in long-term cultures of donors with in vivo exposure to gamma-radiation. No dose-response was apparent. However, in contrast with previous findings, signs of chromosomal instability were observed also in long-term cultures from non-exposed controls. Further studies are needed to assess possible inter-individual differences in the induction of chromosomal instability.

Adolescent↗

Chromosomal instability in human lymphocytes after low dose rate gamma-irradiation and delayed mitogen stimulation.

PURPOSE: To study the possibility that radiation induced chromosomal instability in human lymphocytes is promoted by a conflict between mitogen-induced growth stimulation and radiation-induced genotoxic stress. MATERIALS AND METHODS: Peripheral blood lymphocytes were exposed to low LET-irradiation at: (1) low-dose rate (LDR, 1-3 Gy, 0.024 Gy h[-1]) in order to minimize genotoxic stress; (2) high dose rate (HDR, 1-3 Gy, 45 Gy h[-1]) followed by immediate mitogen stimulation; and (3) HDR followed by a recovery period of 5 days before mitogen stimulation. Subsequent analyses included cell viability and clonogenic cell survival, chromosome aberrations at the first post-irradiation mitosis, and karyotype analysis of long term cultured cells, 11-57 days after mitogen stimulation. RESULTS: Dose (1-3 Gy) and dose rate (LDR and HDR) effects on the frequency of dicentric chromosomes at the first post-irradiation mitosis were in agreement with published data, with a pronounced dose rate effect of 2 and 3 Gy exposures. G-handed karyotypes after 11 days of growth in vitro showed increased frequencies of chromosome breaks and rearrangements in all irradiated cell cultures. Clones with complex karyotype abnormalities and increased frequencies of de novo aberrations developed in the irradiated cultures during extended growth for 22-57 days. These results show that: (1) LDR-irradiation induces chromosomal instability in primary human lymphocytes; (2) mitogen stimulation rescues HDR-irradiated cells from death at the expense of an increased level of chromosome aberrations; and (3) HDR-irradiated cells that are allowed 5 days of recovery before mitogen stimulation develop chromosomal instability during subsequent long-term proliferation. CONCLUSIONS: Neither the acute genotoxic stress of HDR-irradiation compared with LDR-irradiation, nor the hypothesized conflict between mitogen-induced growth stimulation and irradiation-induced growth arrest, seem to be critical conditions for the development of chromosomal instability in primary human T lymphocytes. Post-irradiation incubation allowing apoptotic processes to remove damaged cells does not prevent the subsequent development of chromosomal instability during long-term cell proliferation.

Cells, Cultured↗

Expression and regulation of galanin-R2 receptors in rat primary sensory neurons: effect of axotomy and inflammation.

Using in situ hybridization, we studied galanin-R2 receptor (GAL-R2-R) mRNA in rat lumbar 5 dorsal root ganglia (DRGs) at different time points after peripheral tissue inflammation and sciatic nerve transection (axotomy). About 25% of all normal DRG neuron profiles were GAL-R2-R mRNA-positive, and the majority was of the small type. In normal DRGs GAL-R2-R mRNA often (approximately 80%) colocalized with CGRP mRNA and sometimes (approximately 20%) with GAL-R1-R mRNA. There was a strong increase in the number and labeling intensity of GAL-R2-R mRNA-positive neuron profiles after peripheral tissue inflammation with a peak at 3 days, as well as a long-lasting decrease after axotomy. These results, together with the previously shown regulation of GAL and the GAL-R1-R, suggest that GALergic mechanisms participate in complex adaptive responses in DRGs after inflammation and nerve injury.

Animals↗

Expression and regulation of the neuropeptide Y Y2 receptor in sensory and autonomic ganglia.

The Y2 subtype of neuropeptide tyrosine (NPY) receptors (Y2R) and some neuropeptides have been studied with in situ hybridization in sensory and autonomic neurons of rat and monkey. Between 10% and 20% of the lumbar dorsal root ganglion (DRG) neuron profiles (NPs) contain Y2R mRNA in the rat and monkey. In rat DRGs Y2R mRNA is expressed in calcitonin gene-related peptide (CGRP)-positive, medium-sized, and large neurons, that is in a complementary fashion to the Y1R that is located in small CGRP neurons. In monkey DRGs Y2R mRNA is expressed mainly in small neurons. Peripheral axotomy up-regulates the Y2R in small and large DRG neurons in both species. Y2R and NPY mRNAs are colocalized in many large neurons in axotomized rat DRGs. Y2R mRNA is expressed in 50% of the NPs in the nodose ganglion with a modest increase after axotomy. Y2R mRNA is detected in a few NPs in normal rat superior cervical ganglia, with a marked increase after transection of the carotid nerves. No Y2R mRNA-positive, but many (approximately 30%) weakly Y1R mRNA-positive NPs were found in the sphenopalatine ganglion. Finally, Y2R mRNA levels increase in rat spinal motoneurons after axotomy. Thus, under normal circumstances NPY may act on Y1 and Y2Rs expressed, respectively, in small and large CGRP-positive DRG neurons in the rat. Y2R may be an important receptor in the viscero-sensory neurons. Y2Rs may be particularly important after axotomy serving as presynaptic and/or autoreceptors on rat DRG, superior cervical ganglion, and nodose ganglion neurons and as presynaptic receptors in monkey DRG neurons.

Animals↗

Nitric oxide synthase, choline acetyltransferase, catecholamine enzymes and neuropeptides and their colocalization in the anterior pelvic ganglion, the inferior mesenteric ganglion and the hypogastric nerve of the male guinea pig.

By the indirect immunofluorescence method, the distribution of nitric oxide synthase (NOS)-like immunoreactivity (LI) and its possible colocalization with neuropeptide immunoreactivities, with two enzymes for the catecholamine synthesis pathway, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH), as well as the enzyme for the acetylcholine synthesis pathway, choline acetyltransferase (ChAT) were studied in the anterior pelvic ganglion (APG), the inferior mesenteric ganglion (IMG) and the hypogastric nerve in the male guinea pig. The analyses were performed on tissues from intact animals, as well as after compression/ligation or cut of the hypogastric nerve. In some cases the colonic nerves were also cut. Analysis of the APG showed two main neuronal cell populations, one group containing NOS localized in the caudal part of the APG and one TH-positive group lacking NOS in its cranial part. The majority of the NOS-positive neurons contained ChAT-LI. Some NOS-positive cells did not contain detectable ChAT, but all ChAT-positive cells contained NOS. NOS neurons often contained peptides, including vasoactive intestinal peptide (VIP), neuropeptide tyrosine (NPY), somatostatin (SOM) and/or calcitonin gene-related peptide (CGRP). Some NOS cells expressed DBH, but never TH. The second cell group, characterized by absence of NOS, contained TH, mostly DBH and NPY and occasionally SOM and CGRP. Some TH-positive neurons lacked DBH. In the IMG, the NOS-LI was principally in nerve fibers, which were of two types, one consisting of strongly immunoreactive, coarse, varicose fibers with a patchy distribution, the other one forming fine, varicose, weakly immunoreactive fibers with a more general distribution. In the coarse networks, NOS-LI coexisted with VIP- and DYN-LI and the fibers surrounded mainly the SOM-containing noradrenergic principal ganglion cells. A network of ChAT-positive, often NOS-containing nerve fibers, surrounded the principal neurons. Occasional neuronal cell bodies in the IMG contained both NOS- and ChAT-LI. Accumulation of NOS was observed, both caudal and cranial, to a crush of the hypogastric nerve. VIP accumulated mainly on the caudal side and often coexisted with NOS. NPY accumulated on both sides of the crush, but mainly on the cranial side, and ENK was exclusively on the cranial side. Neither peptide coexisted with NOS. Both substance P (SP) and CGRP showed the strongest accumulation on the cranial side, possibly partly colocalized with NOS. It is concluded that the APG in the male guinea-pig consists of two major complementary neuron populations, the cholinergic neurons always containing NOS and the noradrenergic neurons containing TH and DBH. Some NOS neurons lacked ChAT and could represent truly non-adrenergic, non-cholinergic neurons. In addition, there may be a small dopaminergic neuron population, that is containing TH but lacking DBH. The cholinergic NOS neurons contain varying combinations of peptides. The noradrenergic population often contained NPY and occasionally SOM and CGRP. It is suggested that NO may interact with a number of other messenger molecules to play a role both within the APG and IMG and also in the projection areas of the APG.

Animals↗

Fluticasone propionate aqueous nasal spray in the treatment of nasal polyposis.

BACKGROUND: Topical glucocorticoids are the medical treatment of choice in a majority of patients suffering from nasal polyposis. Fluticasone propionate is a fluorinated steroid reported to be highly effective when used topically in the nose for seasonal and perennial allergic and nonallergic rhinitis. OBJECTIVES: To evaluate the efficacy and tolerability of intranasal fluticasone propionate in the treatment of long-standing polyposis. METHODS: Fifty-five patients with long-standing nasal polyposis were treated over a 26-week period with fluticasone propionate aqueous nasal spray 200 micrograms bid, beclomethasone dipropionate aqueous nasal spray 200 micrograms bid or placebo, administered intranasally in an aqueous spray in a double-blind, placebo-controlled parallel-group design at a single center. The primary efficacy endpoint was the physicians' assessment of symptoms and polyp score. Peak nasal inspiratory flow was performed twice daily and on every visit to evaluate the effect of the corticosteroids on nasal air flow. RESULTS: A significant difference in the primary efficacy endpoint between fluticasone propionate aqueous nasal spray and beclomethasone dipropionate aqueous nasal spray compared with placebo was seen after 14 weeks of treatment. This was further verified by the peak nasal inspiratory flow results. There was some evidence of earlier onset in the fluticasone propionate aqueous nasal spray group compared with the beclomethasone dipropionate aqueous nasal spray group after 4 weeks in terms of the primary efficacy endpoint. From the daily record cards patients receiving fluticasone propionate aqueous nasal spray had a significantly higher percentage of days on which they required no rescue medication (P < .009) and a higher percentage of days with an overall nasal blockage score on waking of < 2 (P < .013) when compared with placebo-treated patients. No other statistically significant results were found between the two active compounds. CONCLUSION: Fluticasone propionate aqueous nasal spray 200 micrograms bid and beclomethasone dipropionate aqueous nasal spray 200 micrograms bid are effective in treating the symptoms of nasal polyps, with some evidence that fluticasone propionate aqueous nasal spray has a faster onset of action and is tolerated at least as well as beclomethasone dipropionate aqueous nasal spray at the same dose.

Administration, Intranasal↗

Comparative analysis of genetic variability among Candida albicans isolates from different geographic locales by three genotypic methods.

The objective of the present study was to conduct a comparative genotypic analysis of Candida albicans isolates from the United States, Europe, and Southeast Asia to determine whether differences between isolates might be associated with geographic locations. The genotypes of 86 unrelated isolates of C. albicans (from the United States and Europe) and 26 isolates from Singapore were examined by three DNA typing methods. Computer-assisted methods were used to analyze the gel patterns for all isolates. A dendrogram based on the overall similarity of the patterns obtained by restriction endonuclease analysis (REA) with EcoRI clustered the U.S. and European isolates into two major groups (groups A and B). The Singaporean isolates demonstrated unique REA profiles, with nine isolates having both or neither of the REA-characteristic 3.7- and 4.2-kb bands present in groups A and B. By REA profiles, the Singaporean isolates were related to each other with similarity values (S(AB)s) of > 0.80, but only one isolate mixed with the U.S. and European isolates at this S(AB) (an arbitrary threshold for genetic similarity). Randomly amplified polymorphic DNA (RAPD) analysis generated DNA profiles that clustered the C. albicans isolates into approximately the same number of distinct typing groups as REA. However, isolates identical to each other by REA were generally different from each other by RAPD analysis. In a composite dendrogram prepared from the results obtained by RAPD analysis, the isolates from the United States and Europe clustered in major groups with S(AB)s of > 0.85, while Singaporean isolates connected to these clusters at S(AB)s of > or = 0.75. Pulsed-field gel electrophoresis was less discriminatory, discerning about one-third as many distinct subtypes as REA or RAPD analysis; the Singaporean isolates were distributed randomly with the U.S. and European isolates. These results suggest that a high degree of genetic diversity exists between C. albicans isolates from Southeast Asia and those from the United States and Europe.

Candida albicans↗

PACAP in the adrenal gland--relationship with choline acetyltransferase, enkephalin and chromaffin cells and effects of immunological sympathectomy.

Using indirect immunohistochemistry and immunological sympathectomy pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity (LI) was studied in the adult rat adrenal gland. All PACAP-positive fibres contained choline acetyltransferase (ChAT)-LI and were found in high numbers among noradrenaline chromaffin cells, whereas enkephalin (ENK)/ChAT-immunoreactive (IR) fibres predominantly innervated adrenaline chromaffin cells. After immunological sympathectomy no PACAP-, ChAT- or ENK-IR fibres remained, strongly suggesting a preganglionic origin. A small number of PACAP-IR fibres was also observed in the subcapsular regions both in controls and in sympathectomized animals, presumably representing sensory fibres. These results define a subpopulation of PACAP-containing cholinergic preganglionic fibres in the adult rat adrenal medulla lacking ENK and innervating noradrenaline chromaffin cells. PACAP was also expressed in a few adrenaline chromaffin cells after immunological removal of the preganglionic innervation, suggesting an additional, hormonal role.

Adrenal Glands↗

Effects of peripheral axotomy on neuropeptides and nitric oxide synthase in dorsal root ganglia and spinal cord of the guinea pig: an immunohistochemical study.

The effect of axotomy (3, 10 and 21 days) on the expression of some neuronal markers was analysed in dorsal root ganglia and spinal cord of guinea-pigs using immunohistochemistry. Three weeks following injury, substance P-like immunoreactivity (-LI) was slightly reduced in the DRGs of the ipsilateral side, whereas a marked increase in neuropeptide Y(NPY)-LI could be detected ipsilaterally and a smaller increase contralaterally. NPY-LI was mainly expressed in small, but also some medium-sized and large neuron profiles after axotomy. Galanin-LI showed a moderate bilateral increase. No significant changes could be observed in DRGs for calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide-, peptide histidine isoleucine- or nitric oxide synthase-LIs. In the ventral horn CGRP-LI was slightly increased bilaterally in motoneurons, most pronounced on the injured side. Autotomy behaviour was seen in seven of the nine animals in the twenty-one day group. The present results demonstrate that also in guinea-pigs several peptides undergo distinct changes in their expression after peripheral nerve injury. However, in contrast to rats and monkeys, galanin-LI is only moderately increased in guinea-pigs. Neuropeptide Y showed a dramatic increase mainly in small neurons, in contrast to the upregulation in large neurons in the rat. Thus, distinct species differences exist with regard to the cellular response to nerve injury.

Animals↗

Effects of Hydrophilization and Immobilization on the Interfacial Behavior of Immunoglobulins.

The adsorption and immobilization of rabbit anti-human immunoglobulin (rabbit IgG), as well as the effects of rinsing with buffer and addition of bovine serum albumin (BSA) or human IgG on the amount and reactivity of bound rabbit IgG, were investigated with ellipsometry, total internal reflection fluorescence spectroscopy (TIRF), and enzyme immuno assay (EIA). It was found that although rabbit IgG readily adsorbs at hydrophobic hexamethyldisiloxane (HMDSO) plasma polymer surfaces, a substantial fraction of the adsorbed protein molecules is desorbed upon rinsing with buffer. BSA was found to adsorb readily at the surfaces obtained after rinsing, although also this protein desorbed to a large extent (about 60%) upon further rinsing with buffer. The adsorption of BSA causes a further reduction in the amount of rabbit IgG adsorbed. Immobilization of rabbit IgG to acrylic acid (AA) plasma polymer surfaces, achieved by covalent coupling via a strongly adsorbed PEG-PEI copolymer, was found to overcome the problem of the desorption of rabbit IgG upon rinsing with buffer or addition of BSA. Furthermore, nonspecific adsorption was virtually absent after immobilization. However, covalently bound rabbit IgG reacted strongly with human IgG, as observed by ellipsometry, TIRF, and EIA. The immobilization of rabbit IgG to hydrophilized surfaces was found to facilitate the interpretation of EIA results.

Journal Article↗

Evaluation of an optimized system for random amplified polymorphic DNA (RAPD)-analysis for genotypic mapping of Candida albicans strains.

A simple, rapid, and cost-effective protocol has been developed for a PCR-based molecular typing method for Candida albicans, which includes the use of a commercially available medium (Chelex 100 Resin) for DNA extraction and a single set of two arbitrarily chosen oligonucleotide (10 nt length) primers for random amplified DNA(RAPD)-analysis. The optimized parameters for the amplification components and conditions for the selected primer combination have been determined to avoid artifactual variation (absence/presence of bands) in RAPD banding patterns in repeated assays. The optimized RAPD-assay consistently generated DNA-patterns of 33 genetically unrelated C. albicans isolates that contained ten polymorphic markers in the non-artifactual banding patterns. The intralaboratory reproducibility of RAPD patterns was efficient and consistent provided the optimized amplification conditions were rigidly controlled. Interlaboratory reproducibility was tempered by slight variations in time of cyclers of different thermocyclers. In comparison, the RAPD assay was almost equal to restriction enzyme analysis (REA) (Eco RI digested chromosomal DNA) in discrimination, and the RAPD assay was able to group isolates of C. albicans that were untypable by REA. The protocol outlined for an optimized RAPD-assay of C. albicans has the potential to be widely useful epidemiological screening tool that can be easily applied in the clinical laboratory.

Candida albicans↗