An HLA-DR11/DQ3 haplotype with a DRB1*0301 sequence motif in the third hypervariable region of the HLA-DR beta-1 chain: molecular and serological analysis of its generation in a European Caucasian family.

The formation of a new human leukocyte antigen (HLA)-DRB1 allele (DRB1*0340) has been detected during the routine testing of a European Caucasian blood and potential stem cell donor and his family. HLA typing of the donor with two polymerase chain reaction - sequence specific oligonucleotides (PCR-SSO) systems yielded inconclusive results. HLA typing of the family members including sequence-based typing of DRB1 in both directions after haplotype-specific amplification showed that the allele had most likely formed by a double crossover event in exon 2 of the DRB1 gene. The HLA haplotype containing the new allele was most probably derived from the father, who was typed as HLA-DRB1*0301,*1101 and DRB3*0101,*0202. The comparison of the sequences of the paternal DRB1 and DRB3 alleles with the exon 2 sequence of the DRB1*0340 showed that it had most likely formed through an uptake of at least the sequence part codons 58-77 of DRB1*0301 (donor) by DRB1*1101 (acceptor). We suppose that the recombination sites are located in the sequences from codons 38-57 and codons 78-88. At the protein level, more than 50% of the alpha-helical structure of the DRB1*1101 chain is replaced by a DRB1*0301-derived sequence with the exchange of several amino acids. Serological typing of the allele showed HLA-DR3. However, one monoclonal anti-DR11 of five DR11-reactive antibodies reacted positive, which might indicate residual immunogenic epitopes of DRB1*1101. HLA alleles that are most similar to HLA-DRB1*0340 are DRB1*030501, *0317, *0329 and *1107 with at least four amino acid differences in exon 2. In conclusion, HLA-DRB1*0340 is a new allele with unique properties compared with other known HLA-DRB alleles with regard to antigenicity, T-cell receptor-binding and peptide-binding possibilities.

Technical note: a new facility for continuous respiration measurements in lactating cows.

An open-circuit indirect calorimetry system consisting of 4 climate-controlled respiration chambers for cattle has been constructed and validated. The system allows for the continuous monitoring of O(2), CO(2), and CH(4) concentrations in chamber air, and the simultaneous determination of feed and water intake, overall physical activity, position changes, standing and lying times, and animal behavior. For complete balance trials, feces, urine, and milk can be collected quantitatively. Most importantly, lactating cows can be milked in the chamber, and blood samples can be drawn from permanent catheters without disruption of the measurements. The investigator, on entering the chamber, wears a facemask connected to the ambient air during the whole milking process. Data are routed to a data acquisition system with appropriate data evaluation software developed in our research unit. Thus, dynamic changes of the above-named parameters during the course of the day or of longer time periods can be monitored. Such data are critical for understanding the complex regulation and interplay of feed intake, energy metabolism, climatic conditions, and milk production.

Numerical modelling of conductive and convective heat transfers in retinal laser applications.

The control of the temperature increase is an important issue in retinal laser treatments. Within the fundus of the eye heat, generated by absorption of light, is transmitted by diffusion in the retinal pigment epithelium and in the choroid and lost by convection due to the choroidal blood flow. The temperature can be spatially and temporally determined by solving the heat equation. In a former analytical model this was achieved by assuming uniform convection for the whole fundus of the eye. A numerical method avoiding this unrealistic assumption by considering convective heat transfer only in the choroid is used here to solve the heat equation. Numerical results are compared with experimental results obtained by using a novel method of noninvasive optoacoustic retinal temperature measurements in rabbits. Assuming global convection the perfusion coefficient was evaluated to 0.07 s(-1), whereas a value of 0.32 s(-1)--much closer to values found in the literature (between 0.28 and 0.30 s(-1))--was obtained when choroidal convection was assumed, showing the advantage of the numerical method. The modelling of retinal laser treatment is thus improved and could be considered in the future to optimize treatments by calculating retinal temperature increases under various tissues and laser properties.

[Solar retinopathy: follow-up examinations with optical coherence tomography (OCT) and microperimetry]. / Retinopathia solaris: Verlaufsbeobachtung mit optischer Kohärenztomografie (OCT) und Mikroperimetrie.

BACKGROUND: Solar retinopathy is a phototoxic damage to the central retina. It may cause visual deterioration and a central scotoma with reading difficulties, that usually fade with time. Morphological and functional changes can be very subtle and are often inadequately detected by standard examinations. PATIENTS AND METHODS: The more affected, left eye of a 41-year-old female patient was examined clinically, by optical coherence tomography (OCT 3) and by MP 1-microperimetry during a 6-month follow up after gazing at a partial eclipse. RESULTS: At all times visual acuity was reduced to 0.7 causing visual deterioration. OCT examinations showed a subfoveal damage of decreasing extent, which was still detectable after 6 months. The retinal affection resulted in a persistent microscotoma of decreasing depth. CONCLUSION: OCT 3 and MP 1-microperimetry are able to detect the morphological and the functional damages in solar retinopathy at a level of sensitivity good enough to produce correlating results which allow the detection of subtle changes during follow-up.

[Selective retina therapy: methods, technique, and online dosimetry]. / Selektive Retinatherapie: Methodik, Technik und Online-Dosimetrie.

Selective retina therapy (SRT) is currently under evaluation, as a new and very subtle laser method, for the treatment of retinal disorders associated with a degradation of the retinal pigmentary epithelium (RPE). SRT makes it possible to selectively effect the RPE, sparing the adjacent neural retina with the photoreceptors and also the choroid below the RPE. In the best case, the therapy leads to regeneration of the RPE and a long-term metabolic increase at the chorio-retinal junction. In contrast to conventional laser photocoagulation, which is associated with complete thermal necrosis of and around the treated site, absolutely no scotoma occurs in SRT. This paper reviews the methods and mechanisms behind the selective effects of the RPE. In vitro and preclinical results are used to describe the bandwidth of selective effects with respect to different irradiation settings. An optoacoustic technique is introduced to visualize effects that cannot be seen by ophthalmoscopy and to facilitate dosimetry control without recourse to angiography completes the report.

[Selective retina therapy in central serous chorioretinopathy with detachment of the pigmentary epithelium]. / Selektive Retina-Therapie bei Retinopathia centralis serosa mit Pigmentepithelabhebung.

BACKGROUND: Selective Retina Therapy (SRT) is a new and innovative laser treatment modality that selectively treats the retinal pigmentary epithelium while sparing the photoreceptors. This therapeutic concept appears to be particularly suitable for treating patients with acute or chronic central serous chorioretinopathy (CSC). We present preliminary results obtained in five patients who had CSC associated with pigmentary epithelium detachment (PED) and serous subretinal fluid (SRF) and who were treated with SRT. METHODS: This case series was made up of five male patients (mean age 47 years) with chronic CSC and SRF resulting from PED. Examinations performed before and at 1 month and 3 months after the treatment were: BCVA, FLA, OCT (Zeiss OCT III). For SRT, confluent treatment of the PED (area of leakage) was carried out using a pulsed frequency-doubled, Q-switched Nd-YLF prototype laser (lambda=527 nm, t= 1.7 s, 100 Hz, energy = 150-250 J). RESULTS: Best corrected visual acuity at baseline was 0.53, while after 4 weeks it was 0.56 and after 12 weeks, 0.5. At baseline leakage was seen at the PED on fluorescein angiography in all patients. After 4 weeks leakage activity was no longer noted on angiography in 4 of 5 patients. OCT at baseline showed SRF at the edge of the PED in all patients, but in 4 of the 5 patients this was no longer detectable after 4 weeks. CONCLUSION: SRT is a safe and effective treatment for patients with CSC in which PED has caused SRF. Not a single case of rip syndrome was observed in this study, even though the PED was treated confluently. Since SRT spares the photoreceptors it is particularly suitable for the treatment of CSC, especially when the origin of leakage is located close to the fovea. The results indicate that SRT leads to reconstruction of the outer blood-retina barrier.

[Selective retina therapy in patients with diabetic maculopathy]. / Selektive Retina-Therapie bei Patienten mit diabetischer Makulopathie.

Selective Retina Therapy (SRT) is a new laser treatment that selectively targets the retinal pigmen epithelium (RPE). In this study, we treated 39 patients presenting with nonischemic, focal and focal-diffuse diabetic maculopathy with SRT. In the main. the results indicate that SRT had stabilizing effects on visual acuity, angiographic leakage, lipid exudation, and foveal retinal thickness. SRT is safe and is especially useful for treating pathologies that are located close to the fovea, which cannot be treated with conventional argon laser photocoagulation.

Selective retina therapy in patients with central serous chorioretinopathy.

BACKGROUND: Central serous chorioretinopathy (CSC) is a disease with a localized breakdown of the outer blood-retinal barrier located within the retinal pigment epithelium (RPE) causing subretinal fluid accumulation. Selective retina therapy (SRT) is a new, minimally invasive laser technology that has been designed to selectively target the RPE. SRT spares retinal tissue. METHODS: Twenty-seven eyes of 27 patients with active CSC were treated with SRT using a pulsed double-Q-switched Nd-YLF prototype laser (lambda=527 nm, t=1.7 micros). At baseline, best-corrected visual acuity was determined and fluorescein angiography and optical coherence tomography were performed. The patients were followed for up to 3 months. RESULTS: After 4 weeks 85.2% of patients showed complete resolution of subretinal fluid and in 96.3% there was no leakage visible on fluorescein angiography. After 3 months 100% of patients demonstrated no subretinal fluid and 100% of patients had no leakage activity on fluorescein angiography. Visual acuity, 20/40 at baseline, improved to 20/28 after 4 weeks and to 20/20 after 3 months. CONCLUSION: SRT is a safe and effective treatment for active CSC. Especially if the RPE leak is located close to the fovea, SRT is the favoured therapeutic option. We recommend earlier treatment of patients with acute CSC in order to prevent development of chronic changes due to CSC with irreversible anatomical and functional damage. SRT might be considered as a first-line treatment for active CSC.

Topographic angiography and optical coherence tomography: a correlation of imaging characteristics.

PURPOSE: Topographic angiography (TAG) using confocal scanning laser angiography and optical coherence tomography (OCT) are new imaging modalities that have been introduced during recent years. OCT and TAG imaging were compared to specify the characteristics of each imaging modality. METHODS: TAG using fluorescein angiography (FA) provides a three-dimensional profile of the vascular structures based on the analysis of a set of 32 confocal images over a depth of 4 mm. OCT provides cross-sectional images of the neurosensory retina and the retinal pigment epithelium-choriocapillary complex (RPE-CC). The authors compared and evaluated both modalities in 10 patients with predominantly classic choroidal neovascularization (CNV), 10 patients with serous pigment epithelial detachment (PED), and 10 patients with geographic RPE atrophy, all secondary to age-related macular degeneration (ARMD). RESULTS: In patients with classic CNV, TAG detected neovascular structures and delineated their configuration. In PEDs pooling of extravascular fluid is demonstrated, and in geographic RPE atrophy TAG showed reduced choroidal perfusion. Classic CNV was demonstrated by OCT as a hyperreflective band at the level of the RPE-CC, and PED showed a dome-shaped RPE detachment. In geographic RPE atrophy, OCT imaged loss of the RPE band and had an increased depth resolution. CONCLUSIONS: TAG and OCT are useful imaging modalities in the evaluation of ARMD cases. TAG visualizes the vascular configuration and dynamic perfusion and leakage changes. OCT is able to document intra-, subretinal, and sub-RPE fluid accumulation secondary to CNV. Both modalities may provide further valuable insight into ARMD pathogenesis, enhance diagnostic quality, and improve the assessment of therapeutic effects.

Immunogenetics of HLA null alleles: implications for blood stem cell transplantation.

The transplantation of haematopoietic stem cells is a potentially curative therapy for a variety of haematological and non-haematological diseases. Matching of donor and recipient for human leucocyte antigens (HLA) is pivotal for the success of blood stem cell transplantation. HLA null alleles are characterized by the lack of a serologically detectable product. Because serological HLA diagnostics are increasingly replaced by DNA-based typing methods considering only small regions of the genes, null alleles may be misdiagnosed as normally expressed variants. The failure to identify an HLA null allele as a non-expressed variant in the stem cell transplantation setting may result in an HLA mismatch that is highly likely to stimulate allogeneic T cells and to trigger graft-vs-host disease. For some HLA null alleles, the translation into a truncated polypeptide chain seems possible, which thus might act as minor histocompatibility antigens. Because the prevalence of HLA null alleles may be around 0.3% or even higher, a screening strategy for HLA null alleles should, therefore, be implemented in the clinical laboratory. It may consist of the combination of serology and standard molecular typing techniques. As the standard molecular techniques are sometimes troublesome especially for characterizing the cytosine island at the 5' end of HLA class I exon 4 and need continuously be updated, an alternative approach may consist of sequencing all samples from genomic DNA for exons 2-3 or 4 (class I) or exon 2 (class II), including the adjacent intron splicing sites. This approach will detect 36/40 so far known non-expressed variants and has the potential to easily uncover novel variants, thus essentially minimizing the risk of overlooking these challenging variants.

Eight novel MICB alleles, including a null allele, identified in gastric MALT lymphoma patients.

MICA and MICB, as members of the major histocompatibility complex (MHC) class I-chain-related genes (MIC), encode stress-inducible glycoproteins that act as activating ligands for NKG2D and gammadelta T-cell receptor-bearing cells. We here describe the identification of eight novel MICB variants, including a null allele, which were identified in peripheral blood leukocytes of gastric MALT lymphoma patients. Only two of the novel alleles are characterized by point mutations, whereas the other variants display a recombination of known exonic MICB sequences that may be best explained by intragenic conversions. The novel MICB null allele is characterized by a Cytosin (C) deletion in a stretch of four Cs beginning from nucleotide 135 of exon 2 that leads to a premature stop codon (TGA) at codon 66.

Impact of tobacco smoking on platelet function in apheresis products in vitro.

BACKGROUND AND OBJECTIVES: The aim of this study was to analyse the platelet function, over a 5-day time-period, of apheresis-derived platelet concentrates obtained from smokers and non-smokers. MATERIALS AND METHODS: Smoker and non-smoker plateletpheresis products were investigated on days 1, 3 and 5 of storage. Receptor expression (as evaluated by flow cytometry) and the platelet aggregation response were measured. RESULTS: There was only a slight loss of platelet function in apheresis products from smokers compared to non-smokers. CONCLUSIONS: Smoking does not significantly change the quality of platelet preparations. The current practice, of not to exclude smokers from platelet donation, can be continued.

Differential platelet receptor expression following hydroxyethyl starch infusion in thrombocytopaenic orthotopic liver transplantation recipients.

BACKGROUND AND OBJECTIVE: Platelet function abnormalities influence the haemostatic defect in patients with liver failure. Patients after orthotopic liver transplantation present thrombocytopaenia associated with bleeding problems, which may be aggravated by the interaction of hydroxyethyl starches with platelets. METHODS: From 12 patients after liver transplantation venous blood samples (3 mL) were taken before, 20 and 120 min after infusion of hydroxyethyl starch of medium molecular weight (200 kDa/0.5) 6% 10 mL kg(-1) over a period of 30 min. Surface expression of glycoprotein IIb/IIIa and P-selectin were quantified by flow cytometry as well as the percentage of platelet-leucocyte complexes. RESULTS: A significant decrease of P-selectin expression following administration of hydroxyethyl starch after 120 min (89.1 +/- 4.2%, P = 0.029) and a corresponding significant reduction in the formation of platelet-monocyte complexes (81.1 +/- 7.8%, P = 0.001) were observed. There was no alteration in the glycoprotein IIb/IIIa expression after hydroxyethyl starch infusion. CONCLUSIONS: Infusion of hydroxyethyl starch 200 kDa/0.5 in clinically relevant doses does not alter glycoprotein IIb/IIIa expression in thrombocytopaenic patients with pre-existing platelet dysfunction after orthotopic liver transplantation. Accordingly, infusion of hydroxyethyl starch may have a beneficial effect on microvascular graft perfusion through the resulting haemodilution and reduced P-selectin expression with subsequent reduced leucocyte-platelet complexes and endothelial adhesion.

The phylogenies of introns 4-7 demonstrate an inconsistent pattern between human leukocyte antigen-C group topologies.

In this study, we have sequenced introns 4-7 in 31 human leukocyte antigen-C (HLA-C) alleles representing all allelic groups. Intron sequences show a patchwork pattern of polymorphism. Bootstrap support for phylogenetic lineages and for differentiation between groups is limited due to the high homology of intron sequences, where the substitution of a single nucleotide may lead to the assignment to different clusters. The intron data suggest the idea of a Cw*06/Cw*12 family, which is closely related to a hypothetical Cw*05/Cw*08 family. Moreover, a third family consisting of Cw*01/Cw*04/Cw*18 may exist. The intron data compiled in our study may be the basis for further sequencing studies. The detection of three novel alleles (Cw*0401new, Cw*140201new, and Cw*1701new) suggests that the HLA-C polymorphism might have been strongly underestimated to date.

HistoCheck: rating of HLA class I and II mismatches by an internet-based software tool.

HLA polymorphism is a major barrier for hematopoietic stem cell and solid organ transplantation. To estimate the allogeneic potential between HLA-mismatched stem cell donor/recipient pairs, we recently proposed a matching score (dissimilarity index) that is based on the structural data of HLA class I molecules, and on the functional similarity of amino acids (AA). This first approach revealed new features about presumptive subtype allogenicities within the HLA-A*23 and A*24 groups. We have now developed an internet-based software tool ("HistoCheck") that is capable to assess the allogenicity (matching score) between any pair of clinically relevant HLA class I, and also class II, alleles. Newly described HLA sequences will be regularly integrated into the database according to the nomenclature for factors of the HLA system updates. The software is intended to be a first step for estimating the allogenicity of HLA mismatches in peculiar clinical settings, as long as there are no reliable in vitro or clinical studies available. The algorithm can later be modified according to functional data, for example, peptide-binding specificities. With the extension of the sequence similarity concept to all clinically relevant HLA class I and II loci, HistoCheck may contribute to prevent HLA mismatching being a matter of chance.

Postictal serum levels of antiepileptic drugs for detection of noncompliance.

Medication noncompliance (NC) is thought to be a major cause of insufficient seizure control. In an explorative study we investigated whether postictal serum levels (PISLs) of antiepileptic drugs (AEDs) are a reliable indicator of NC. Young adults with epilepsy on a stable AED regimen were asked to contact medical service as soon as possible when a seizure occurred to obtain serum levels of their AEDs. PISLs were compared with the mean value of two routine serum levels of the same medication. PISLs lower than 50% of the individual reference value were regarded as an indicator of NC. PISL samples in 61 seizures of 52 patients treated mainly with carbamazepine, valproic acid, or lamotrigine were evaluated. A drop in serum levels >50% indicating NC was noted in 44.3% of the seizures. Determination of PISLs seems to be a simple and useful method for detecting or ruling out irregular intake patterns as a cause of "breakthrough" seizures.

The nature of introns 4-6 suggests reduced lineage specificity in HLA-B alleles.

For most HLA-B alleles, coding sequences of the 3' part of the genes still need to be determined, and sequences of the 3' noncoding regions have yet to be studied systematically. In this study, we have determined the sequences of introns 4-6 in all HLA-B allelic groups, and computed nucleotide substitution rates and phylogenetic relationships. These sequences demonstrated an inconsistent pattern of intralineage specificity, intralineage diversity, and interlineage diversity that is best characterized by a patchwork pattern. Apart from phylogenetic studies about HLA diversity and diversification, the sequence data obtained in our study may prove valuable for haplotype-specific sequencing of the 3' part of HLA-B and for the explanation of recombination events in newly described HLA-B alleles.

[German diagnosis-related groups. Problems in the field of mental and behavioural disorders shown in the example of hospital treatment of alcoholics]. / German Diagnosis Related Groups. Problemfelder im Bereich psychischer Störungen am Beispiel der Krankenhausbehandlung Alkoholabhängiger.

Hospital treatment in Germany will clearly change: instead of paying by the number of days, blanket prices including all necessary benefits will become the norm. The nearly innumerable treatments possible were collected into diagnosis-related groups (DRGs) requiring similar clinicomedical benefits and consuming similar financial resources. The DRG classification in Germany (G-DRG) will be oriented to the Australian refined diagnosis-related group (AR-DRG) system, which has existed since 1998. German psychiatric hospitals have by policy not yet been involved in the new system. However, a big sector of psychiatric activity--as far as number of patients is concerned--has already been incorporated in this new system: detoxification from legal and illegal drugs is involved in G-DRG inasmuch as it does not take place in psychiatric hospitals. If the DRG system becomes established in German psychiatric hospitals, grave difficulties will occur. These problems are described together with the expected results in the example of hospital treatment of alcoholics.

Lack of toxic side effects in neutrophils following hyperbaric oxygen.

Conflicting data have been reported about the impact of repeated HBO2 exposure on the production of superoxide radicals during the neutrophil respiratory burst (RB) and on phagocytosis. In this study we wanted to see if exposure to hyperoxia would affect human neutrophil RB and phagocytosis. Short- and long-term effects after single or repetitive HBO2 exposure of 2.5 atmospheres absolute over a period of 90 min were studied in 40 healthy volunteers. The RB was measured by the intracellular oxidation of dihydrorhodamine after induction by Escherichia coli (E. coli), or priming with recombinant tumour necrosis factor alpha (TNF-alpha), followed by N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. The phagocytic activity was determined by the intake of FITC-labelled opsonized E. coli. No differences could be found between RB and phagocytic activity before and after HBO2 therapy, regardless of short- or long-term exposure. These findings indicate that exposure to hyperoxia does not impair these two important functions of the human innate host defense.

HLA-B*4431: a new HLA-B variant with a complex ancestral history involving HLA-B*44, B*40 and B*07 alleles.

We here describe the identification of the new allele HLA-B*4431, which was found in three members of a Turkish family. Sequencing of the new allele following haplotype-specific PCR amplification revealed that exon 2 is identical to HLA-B*4402, whereas exon 3 resembles a HLA-B*40 variant with the exception of position 572, where a single nucleotide transversion (C > G) leads to an amino acid exchange (Trp162Ser). The generation of the 3' part of B*4431 may be best explained by a separate recombination between B*40 and B*07. Although B*4431 consists of B44 in its alpha1 domain and of B60(40) in its alpha2 domain; the new allele only displayed B44 seroreactivity, which demonstrates that epitopes crucial for B60(40) specificity must be located in the alpha1 domain.