Cartilage calcification is associated with histological degeneration of the knee joint: a highly prevalent, age-independent systemic process.

OBJECTIVES: To investigate if cartilage calcification (CC) is a systemic process, the purpose of this study was to determine the prevalence and the amount of meniscal/hyaline CC of the knee joint in the general population by high-resolution imaging (DCR) and to evaluate the association between CC with cartilage degeneration and age. METHODS: Cross-sectional DCR-study of 180 knee joints of 90 donors (42 female/48 male, mean age 62.3y). Histological hyaline (OARSI) and meniscal (Krenn) cartilage degeneration was determined of all knees. RESULTS: CC was observed in 100% of the donors (bilaterally in 98%), hyaline cartilage calcification (HCC) in 92% and meniscal calcification (MC) in 100%. CC was detected in more than three out of six distinct cartilage areas in 84.4% of all knees. The mean amount of CC correlated between both sides of donors, the different analyzed areas of the knee joint and between the various types of cartilage structures. There was more calcification in meniscal than in hyaline cartilage (factor 5.3) and in the medial than the lateral compartment (factor 1.2). HCC/MC were already detectable with only mild cartilage lesions and the amount correlated with histological cartilage degeneration, but not with age. CONCLUSIONS: The present study provides evidence that meniscal and hyaline CC occurs in a pattern that is compatible with CC being a systemically driven process and that meniscal fibrocartilage is more prone to calcification than hyaline cartilage. Furthermore, the age-independent association between the amount of CC and the grade of degeneration in both hyaline and meniscal cartilage, suggests that CC is an obligatory early event in initiating cartilage degeneration.

[Refixation of osteochondral fragments with resorbable polylactid implants : Long-term clincal and MRI results]. / Refixation von osteochondralen Fragmenten mit resorbierbaren Polylactid-Implantaten : Klinische und MR-morphologische Langzeitergebnisse.

BACKGROUND: Refixation with resorbable implants is a common surgical treatment in patients who suffer an injury with shearing of an osteochondral flake due to trauma of the knee or the upper ankle joint. To date there are no studies which outline long-term outcomes for this procedure. The aim of this study was to evaluate long-term clinical and magnetic resonance imaging (MRI) results after refixation with resorbable polylactide (PLLA) implants. MATERIAL AND METHODS: In this retrospective study 12 patients with 13 injuries were examined 13.9 years (±1.2 years) after refixation of an osteochondral fragment of the knee (10 patients) and the upper ankle joint (2 patients) with a mean size of 3.33 cm2 (±2.33) by resorbable polylactide (PLLA) implants (nails, pins, screws, Bionx, Tampere, Finland). To objectify the clinical results eight established clinical scores (VASS, Tegner, Lysholm, McDermott, KSS, WOMAC, AOFAS, FADI+Sports) were used. Furthermore, the morphological integration of bone and cartilage was assessed by MRI (3 T) using proton-weighted and cartilage-sensitive 3D double-echo steady-state (DESS) sequences. The morphological results were objectified with a modified MRI score according to Henderson et al. RESULTS: After 13.9 years (±1.2) the patients with an injury of the knee as well as of the upper ankle joint showed good to excellent results (knee: VASS 1.2 (±1.7), Tegner 4.4 (±1.3), Lysholm 85.7 (±12.2), McDermott 90.7 (±8.6), KSS 189 (±14.2), WOMAC (6.16% (±8.45)) (upper ankle joint: VASS 2.5 (±2.5), Tegner 5.5 (±1.5), Lysholm 87 (±13), McDermott 88 (±12); WOMAC (8.54% (±8.54), AOFAS 75.5 (±24.5), FADI+Sports 118 (±18)). In all cases there was evidence of good integration of the osteochondral fragment in MRI. In five patients there was moderate subchondral cyst formation (∅ ≤1 mm); however, mild changes of the cartilage contour were found in all patients. The mean modified Henderson score achieved was 14.4 (±2.0, best 8, worst 32), which corresponds to a good morphological result. CONCLUSION: Because of good clinical and morphological results shown by MRI, refixation through resorbable implants (PLLA) can be recommended for treatment of traumatic osteochondral flakes.

[Promoting Young Talents in Trauma Surgery through Students-On-Call]. / Nachwuchsförderung in der Unfallchirurgie durch studentischen Rufdienst.

BACKGROUND: Due to restrictions on admission to medical school, changing claims to an optimized work-life balance and occupational perspectives, surgical professions in particular are struggling with strategies to motivate young academics. Surgical disziplines aim towards a profound transfer of knowledge and pique student's interest by ensuring a sustainable education at university. OBJECTIVES: The goal of this study was to evaluate a Students-On-Call System (SOCS) and to identify a financial benefit. MATERIALS AND METHODS: In this study the SOCS was compared pre-/postevaluation using questionnaires and the supporting X­rays within a curricular teaching module of orthopedic trauma surgery, with students in the fourth semester of specialism and those in the practical semester at medical school. RESULTS: The students of SOCS showed significantly better results prior to the course and afterwards than the two other groups. By establishing SOCS medical students get involved into the treatment of emergency patients in the trauma resuscitation unit (TRU) and operating room (OR). Students get the chance to enhance their comprehension of diagnostics, therapy and decision making in surgical context. This highly valuable traineeship combines a minimized teaching effort with an effective motivation of young academcis for the surgical profession. A SOCS has reduced the workload of medical colleagues. Establishing SOCS spare the residents being on call and results in reduced costs of 23,659.86 Euro per year. CONCLUSION: The results presented show that the SOCS leads to an excellent cost-benefit balance, which has been established in multiple surgical departments at the medical school of the University of Göttingen. Apart from practice-oriented surgical teaching, the SOCS is a way of promoting successful young talent saving resources in the medical on-call services.

[The treatment of patellar dislocation: a systematic review]. / Die Therapie der Patellaluxation: eine systematische Literaturanalyse.

AIM: The diagnosis and treatment of patellar dislocation is very complex. The aim of this study is to give an overview of the biomechanics of the patellofemoral joint and to point out the latest developments in diagnosis and treatment of patellar dislocation. METHOD: The authors electronically searched Medline, Cochrane and Embase for studies on the biomechanics of the patellofemoral joint and for conservative and surgical treatments after patellar dislocation. We extracted baseline demographics, biomechanical, conservation and surgical details. RESULTS: Understanding the biomechanics of the patellofemoral joint is necessary to understand the pathology of patellar dislocation. The patellofemoral joint consists of a complex system of static, active and passive stabilising factors. Patellar instability can result from osseous and soft-tissue abnormalities, such as trochlear dysplasia, patella alta, a high tibial tuberosity trochlear groove (TTTG) distance, weaknesses of the vastus medialis obliquus or a lesion of the medial retinaculum. Recent studies have focused on the medial patellofemoral ligament (MPFL) and have shown that the MPFL is the most significant passive stabiliser of the patella. Following patellar dislocation, an MRI should be standard practice to detect an MPFL rupture, osteochondral lesions or other risk factors for redislocation. An acute first-time patellar dislocation without osteochondral lesions and without severe risk factors for a redislocation should follow a conservative treatment plan. If surgical treatment is required, the best postoperative results occur when the MPFL is reconstructed, leading to a redislocation rate of 5%, this includes cases that have a dysplastic trochlea. Duplication of the medial retinaculum show very inconsistent results in the literature, possibly due to the fact that the essential pathomorphology of patellar dislocation is not addressed. Addressing the exact location of the rupture of the MPFL with a suture is possibly more convenient, especially after first-time dislocation with associated risk factors for a redislocation. Recent literature does not encourage the use of lateral release, since this can increase patellar instability. Indications for lateral release include persistent patellar instability or pain reduction in an older arthritic subject. For correcting a patellofemoral malalignment, the TTTG distance should be measured and a medial transposition of the anterior tibial tubercle hinged on a distal periosteal attachment should be considered. Cartilage lesions on the medial facet of the patella are a contra-indication for medial tubercle transposition. For cartilage lesions of the lateral facet, antero-medialization of the tibial tubercle can be successful. A tubercle osteotomy can be efficiently combined with MPFL reconstruction. We believe that patients with open epiphyseal plates should be treated with duplication of the medial retinaculum. In the presence of patellar maltracking, an additional subperiostal soft tissue release with medialisation of the distal part of the patellar tendon can be performed. CONCLUSION: It seems that the predominating factors for patellar dislocation are heterogenic morphology in combination with individual predisposition. Non-surgical treatment is typically recommended for primary patellar dislocation without any osteochondral lesions and in the absence of significant risk factors for redislocation. If surgical treatment is deemed necessary, addressing the essential pathomorphology has become the primary focus.

[Mid-term clinical and MRI results after refixation of osteochondral fractures with resorbable implants]. / Mittelfristige klinische und kernspintomografische Ergebnisse nach Refixation osteochondraler Fragmente mit resorbierbaren Implantaten.

AIM: Refixation of osteochondral fractures with resorbable implants is a common surgical treatment. There are almost no studies that prove good clinical outcomes. Hence, the aim of the study was to evaluate the mid-term results after refixation of osteochondral fractures. METHODS: The results of 12 patients were recorded 6.5 (±1) years after refixation of osteochondral fractures measuring 3.4 cm (2) (±2.5) of the knee (8 ×) or the ankle joint (4 ×) with resorbable inplants. Clinical scores and a modified MRI score based on that of Henderson et al. were used. RESULTS: The clinical scores showed good to excellent results after 6.5 (±1) years (VAS pain: 1.9 [±2.4], Tegner: 5.0 [±1.7], Lysholm: 84.8 [±14.3], McDermott: 91.3 [±7.9], Knee Society: 189.4 [±12.1]). MRI showed with one exception good integration of the fractures. In 3 cases subchondral cysts could be found. In 7 cases changes in the chondral outline occurred. The effect of this was a modified Henderson score of 12.6 (±3.7). The MRI results did not correlate with the clinical outcome. CONCLUSION: Because of its good clinical results the refixation with resorbable implants can be recommended to treat osteochondral fractures.

The nodulin vfENOD18 is an ATP-binding protein in infected cells of Vicia faba L. nodules.

Recently we described the novel nodulin gene VfENOD18, whose corresponding transcripts were restricted to the nitrogen-fixing zone III of broad bean root nodules. To characterize VfENOD18 on the protein level, polyclonal antibodies were generated using the purified recombinant VfENOD18 protein produced in Escherichia coli by employing the pMAL-c expression system. These antibodies recognized immunoreactive proteins isolated from indeterminate nodules of different leguminous plants, but also from non-symbiotic tissues of Glycine max and from tissues of Arabidopsis thaliana and Zea mays. Using immunogold labelling the nodulin VfENOD18 was localized to the cytoplasm of infected cells in the nitrogen-fixing zone of broad bean nodules. Due to the homology of the VfENOD18 sequence to that of the ATP-binding protein MJ0577 from the hyperthermophile Methanococcus jannaschii the recombinant VfENOD18 protein was tested for ATP-binding. Using the biotin photoaffinity ATP analogue 8N3ATP[gamma]biotin it could be demonstrated that VfENOD18 is an ATP-binding protein. PCR experiments revealed that the amino acid sequences of the putative C-terminal ATP-binding sites of the VfENOD 18 homologues from Lens culinaris, Vicia hirsuta, Vicia sativa and Vicia villosa were conserved. We propose that VfENOD18 is a member of a novel family of ATP-binding proteins in plants.

The broad bean nodulin VfENOD18 is a member of a novel family of plant proteins with homologies to the bacterial MJ0577 superfamily.

Full-length transcript sequences were isolated from broad bean root nodules, which encode a novel nodulin designated VfENOD18. The corresponding transcripts were detected in early and in late stages of nodule development and were localized exclusively in the nitrogen-fixing zone III. The VfENOD18 sequence is not only homologous to a number of ESTs from various mono- and dicotyledonous plants, but also to the ATP-binding protein MJ0577 from Methanococcus jannaschii and to a range of bacterial proteins that belong to the MJ0577 superfamily. Hence, VfENOD18 is a member of a ubiquitous family of plant proteins that might function as ATP-binding proteins or ATPases. On the genomic level, VfENOD18 genes can be divided into two groups on the basis of differences in their 5' UTRs. One group lacks the 5' UTR region including the ATG initiation codon, whereas the second group contained the complete 5' UTR region. Further upstream of this VfENOD18 gene, a retrotransposon sequence was identified. The -14/-964 VfENOD18 promoter fragment was devoid of complete organ-specific elements known from other nodulin gene promoters. Nevertheless, this region was able to mediate full promoter activity in the central region of transgenic Vicia hirsuta root nodules.

Resistance to Trypanosoma cruzi infection in mice does not necessarily correlate with production of interferon-gamma in vivo.

Interferon-gamma (IFN-gamma) is the most important mediator of inhibition of intracellular replication of Trypanosoma cruzi in vitro and has a protective effect against this parasite if administered in vivo. Here we have analyzed the importance of IFN-gamma for resistance against a lethal infection with T. cruzi in a mouse model system. Resistant B6D2 mice survived the infection with a virulent strain of T. cruzi, whereas susceptible BALB/c mice died within 3 weeks. Both strains produced large amounts of IFN-gamma after infection. Surprisingly, susceptible mice had higher serum concentrations of IFN-gamma and showed, using in situ hybridization a stronger increase in IFN-gamma mRNA-producing cells in their spleens than resistant mice. Moreover, this pattern was also found when immune spleen cells were stimulated with parasite antigens in vitro. However, a marked difference between these mice was found in the production of IL-4, which was much higher in susceptible mice in vivo and in vitro. No difference was found for IL-10. These data show that, at least in the mouse strain/parasite combination used, production of IFN-gamma is not the decisive factor determining resistance or susceptibility to T. cruzi. Rather, it is possible that the balance between protective (e.g., IFN-gamma) and exacerbative cytokines (e.g., IL-4) may decide over disease control or progression.

Expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES genes in lymph nodes from HIV+ individuals: correlation with a Th1-type cytokine response.

The in vivo response of the immune system after HIV infection in regard to cytokine production and C-C chemokine synthesis is not well known. Here we have analysed cytokine and chemokine mRNA production in lymph nodes with follicular hyperplasia (FHLN) of HIV-infected patients by in situ hybridization using anti-sense mRNA probes. The synthesis of mRNAs for interferon-gamma (IFN-gamma), IL-12p35, IL-12p40, IL-4, and for the C-C chemokines RANTES, MIP-1alpha, and MIP-1beta was compared with that of lymph nodes from non-infected individuals to define HIV-specific events. Only few cells expressing IFN-gamma, RANTES, MIP-1alpha, and MIP-1beta mRNAs were detectable in the T-dependent area of lymph nodes from HIV-negatives. In contrast, in FHLN from HIV+ patients a high number of IFN-gamma, RANTES, MIP-1alpha, and MIP-1beta mRNA-containing cells were detectable. Remarkably, only single individual IL-12p35 mRNA-producing cells were present in the T-dependent area from both HIV+ and HIV lymph nodes. Furthermore, the low number of IL-12p40 mRNA-expressing cells did not differ between HIV+ and HIV- lymph nodes. This indicates that IFN-gamma is expressed independently of IL-12, possibly by a direct T cell-mediated reaction. IL-4 mRNA-producing cells were hardly detectable in infected and control lymph nodes. The same findings were made in a limited number of samples from patients with advanced disease. Thus, these results demonstrate that a high IFN-gamma production is accompanied by a strong expression of MIP-1alpha, MIP-1beta, and RANTES in the lymph node after HIV infection. This favours the idea that a Th1-type immune response correlates with a preferential production of C-C chemokines in FHLN of HIV+ patients.

Trypanosoma cruzi induces strong IL-12 and IL-18 gene expression in vivo: correlation with interferon-gamma (IFN-gamma) production.

IFN-gamma, produced after infection with Trypanosoma cruzi, has been shown to be crucial in the determination of resistance or susceptibility. We have performed a detailed study on the expression of IFN-gamma and of the IFN-gamma-inducing cytokines IL-12 and IFN-gamma-inducing factor (IGIF)/IL-18 with regard to time course and tissue localization. IFN-gamma was present in high amounts in the serum and in the supernatants of unseparated spleen cells and isolated CD4+ and CD8+ T cells from the spleens of infected mice which were stimulated ex vivo with T. cruzi. Using the in situ hybridization technique we demonstrate that IL-12 p40 messages were expressed in the spleen and increased during infection, correlating with the expression of IFN-gamma transcripts. Furthermore, we show for the first time that the mRNA for the cytokine IL-18 was induced by a parasitic infection and that this expression increased during infection with T. cruzi. Interestingly, the message for IL-18 was produced earlier during infection and already had declined until day 38, when IFN-gamma and IL-12 p40 transcripts were optimally expressed. Surprisingly, the changes in IL-12 and IL-18 mRNA production were clearly seen only by in situ hybridization, but less clearly by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). This is possibly due to the extensive activation and proliferation of spleen cells observed during infection leading to a dilution of these specific mRNAs.

The long term effects of traumatic brain injury on the roles of caregivers.

This study was conducted to identify the long term effects of traumatic brain injury (TBI) on the roles of caregivers. The subjects consisted of 155 caregivers of survivors with TBI who were randomly selected from 15 midwestern state brain injury association databases. A questionnaire was developed by the researchers to determine factors affecting role changes of caregivers. The Role Checklist, by Barris, Oakley and Kielhofner, was also included with the questionnaire. Both were mailed to each selected caregiver and used for data gathering. The data obtained were analysed to determine existing trends in the data. Graphs were utilized to depict the trends that was identified. The following trends and conclusions established by this research include: (a) behavioural effects of the survivor with a TBI are associated with the number of role changes experienced by caregivers; (b) participation in support systems is associated with the number of role changes experienced by caregivers; and (c) caregivers who care for a person with a TBI in the home will show a larger number of role changes than those who do not provide direct care for a person with a TBI.

Infection with Trypanosoma cruzi selectively upregulates B7-2 molecules on macrophages and enhances their costimulatory activity.

T-cell-mediated immune responses are essential for protection against infection with the protozoan Trypanosoma cruzi. In this study, we investigated the influence of infection of murine macrophages with T. cruzi on costimulatory signals for T lymphocytes provided by these cells. We demonstrate that bone marrow-derived macrophages (BMMph) selectively and strongly upregulate expression of B7-2 molecules after infection, while the expression of other costimulatory molecules such as B7-1, intercellular adhesion molecule 1, lymphocyte function-associated antigen 3, and heat-stable antigen is not significantly affected. Infection by live trypanosomes was required. As a consequence of the strong B7-2 upregulation, the infected macrophages are able to induce proliferation of splenic CD4+ T cells in the presence of anti-CD3 antibodies with much higher efficiency than uninfected macrophages. Costimulation could be inhibited by an antibody to B7-2. Furthermore, costimulatory activity for established T-cell clones of Th1 and Th2 phenotype was also strongly enhanced in BMMph by infection with T. cruzi. Th1 cells stimulated either via anti-CD3 antibodies or via specific antigen proliferated with higher efficiency in the presence of infected macrophages than in the presence of uninfected cells. BMMph stimulated with gamma interferon (IFN-gamma), expressing elevated levels of B7-2 molecules, are also able to enhance Th1 cell proliferation, which was highest, using macrophages which were infected and in parallel were stimulated with IFN-gamma. Noteworthy, for cloned Th2 cells, the mechanism of costimulation differed, because costimulation of Th2 cells was not dependent on B7-2 upregulation but was due to secretion of interleukin-1alpha. These findings demonstrate that infection of macrophages with T. cruzi transforms the macrophage into a potent costimulatory cell based on the induction of two different costimulatory activities.

Modulation of proliferation and lymphokine secretion of murine CD4+ T cells and cloned Th1 cells by proteins of the extracellular matrix.

In this study we investigated the co-stimulatory signaling capacity of diverse proteins of the extracellular matrix (ECM) for murine resting CD4+ T cells and Th1 clone cells, activated by immobilized anti-CD3 mAb. ECM proteins used in various concentrations had no effect on IL-2 production or proliferation of highly purified CD4+ T cell populations. When the preparation of CD4+ T cells contained contaminating accessory cells, IL-2 secretion and proliferation was enhanced in the presence of co-immobilized collagens or fibronectin. However, the level of proliferation attainable by added irradiated splenocytes was not reached. Using Th1 cell clone M4, enhanced production of IL-2 in the presence of immobilized ECM proteins was observed. At a submitogenic anti-CD3 mAb dose, proliferation of M4 T cells was augmented by the ECM proteins in a concentration range that optimally induced IL-2 production. IL-2R p55 was up-regulated on M4 T cells by collagen type IV and fibronectin to the same level that was induced by exogenously added IL-2, whereas added accessory cells induced a higher level of IL-2R p55 expression. Likewise, in dot-blot analysis a comparable quantity of IL-2R p55- and p75-specific transcripts was induced by collagen type IV or fibronectin and by IL-2, which was lower than that induced by antigen-presenting cells. Our data suggest that the enhanced proliferation of M4 T cells induced by ECM proteins is not the consequence of direct up-regulation of IL-2R, but appears to be due indirectly to elevated secretion of IL-2. At an optimal anti-CD3 mAb dose the collagens inhibited M4 T cell proliferation. Diminished cell surface expression of IL-2R p55 following stimulation with anti-CD3 mAb plus collagen type IV compared with anti-CD3 mAb alone was observed and may be responsible for growth inhibition.

Costimulatory signalling potential of murine MHC class II-positive T-clone cells.

Activated human and rat T cells as well as mouse T-cell clones have been reported to synthesize and express major histocompatibility complex (MHC) class II molecules. However, the capacity of class II+ antigen (Ag) presenting T cells to induce proliferation of Ag-specific cloned T cells has been controversial. We analysed whether the failure of some T-cell clones to proliferate in response to Ag presented by class II+ T cells is because of a lack of costimulatory cytokine production by the antigen-presenting cells (APC). As a model system the mouse class II+ cloned BI/O4.1 T cells were used as APC in order to activate the T cell clone KIII5. This T-helper 1 (Th1) type, GAT (synthetic copolymer of L-glutamic acid, L-alanine and L-tyrosine)-specific clone is characterized by an efficient downregulation of interleukin-2 receptor (IL-2R) with time following antigenic stimulation. KIII5 cells respond to GAT-presenting splenic antigen-presenting cells (APC) by IL-2 production, IL-2R upregulation and proliferation. When BI/O4.1 T cells were used as APC, KIII5 cells produced IL-2, but did not proliferate. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed a lack of IL-12 production by BI/O4.1 cells. Addition of IL-12 to a coculture of Ag-presenting BI/O4.1 cells and KIII5 cells fully reconstituted a proliferative response. IL-12 in synergy with IL-2 upregulated IL-2R alpha chain expression and enhanced proliferation of KIII5 cells. Our data suggest, that class II+ T cells are not functional in inducing Ag-mediated expansion of resting Th1 cells owing to their failure to produce IL-12, but rather that they play a role in amplification loops during an ongoing immune response.

Trypanosoma cruzi is a potent inducer of interleukin-12 production in macrophages.

Cytokines produced after infection with Trypanosoma cruzi have been shown to be crucial in the determination of resistance or susceptibility. Interferon-gamma (IFN-gamma) is the predominant cytokine produced after infection and has been shown to protect susceptible mice from infection. IFN-gamma production by natural killer cells and T cells is induced by interleukin-12 (IL-12). Therefore, the aim of our study was to analyze the ability of T. cruzi to induce IL-12 production. Spleen cells and bone marrow-derived macrophages incubated with T. cruzi trypomastigotes induced high amounts of IL-12p40 mRNA as shown by reverse transcriptase-polymerase chain reaction. Lipopolysaccharide (LPS) was less efficient in inducing IL-12p40-specific mRNA. Furthermore, biologically active IL-12, detected by the capacity of the supernatant of infected macrophages to induce IFN-gamma production in spleen cells, was produced at very high levels. In comparison, macrophages stimulated with LPS secreted drastically less IL-12. Interestingly, only live, UV- or gamma-irradiated trypanosomes, but not heat-killed parasites or lysates, were functional in this respect. In a kinetic study, in the supernatant obtained from cultures of infected macrophages, IL-12 was already detectable at 2 h after infection, peaked at 32 h and declined after 45 h.

The broad bean gene VfNOD32 encodes a nodulin with sequence similarities to chitinases that is homologous to (alpha/beta)8-barrel-type seed proteins.

Nodulin gene transcripts isolated from a broad bean (Vicia faba L.) root nodule cDNA library and designated VfNOD32 are detectable in the nitrogen-fixing zone III of nodules and in much smaller amounts in flowers. In nodules, these transcripts are detectable for the first time 7 d after inoculation, at least 1 d before leghemoglobin gene transcription starts. Two putative full-length cDNAs representing different transcript sequences of 92.5% identity were sequenced. The corresponding broad bean genes were termed VfNOD32-A1 and VfNOD32-A2, and the encoded proteins were termed Nvf32-A1 and Nvf32-A2. The derived amino acid sequences of the Nvf32 proteins are highly homologous to the Vicia narbonensis (alpha/beta)8-barrel seed protein narbonin. Considering this homology, Nvf32 is assumed to have a similar structure consisting of beta-sheets forming a central barrel surrounded by alpha-helices. The two Nvf32 sequences also contain two conserved amino acid motifs that are characteristic of class-III chitinases. Several amino acids demonstrated to be essential for chitinase activity are conserved in both regions, whereas one essential glutamic acid was changed to glycine in the Nvf32-A1 isoform but not in the Nvf32-A2 isoform.

The capacity of bone marrow-derived macrophages to process bovine insulin is regulated by lymphokines.

The antigen presentation capacity of bone marrow-derived macrophages (BMMph) was shown previously to be increased after stimulation with the lymphokines IFN-gamma or granulocyte macrophage colony stimulating factor (GM-CSF) respectively. Using bovine insulin (BI) as antigen, activation of BMMph with GM-CSF resulted in the generation of highly effective presenting cells. In contrast, IFN-gamma-treated macrophages, although better presenters than untreated BMMph, stimulated BI-specific T hybridoma cells only weakly to IL-2 production despite the fact that they expressed drastically more MHC class II molecules than GM-CSF-activated BMMph. Therefore we analyzed whether the observed differences in the presentation function of GM-CSF- and IFN-gamma-pulsed BMMph might be a consequence of differences in their capability to process BI. By blocking thiol and serine proteases with specific inhibitors or by raising the intracellular pH with chloroquine during BI pulse, the presentation capacity of IFN-gamma-activated BMMph was significantly enhanced, while the presentation function of GM-CSF-pulsed macrophages was not positively influenced. These findings suggest that the activity of thiol/serine proteases in BMMph is differently influenced by the two cytokines. A regulatory influence of the cytokines on the activity of metallo and acidic proteases was not observed. Thus, the weaker BI presentation capacity of IFN-gamma-treated macrophages as compared with GM-CSF-pulsed cells seems to be the consequence of a more excessive degradation of BI and destruction of the antigenic epitope.

The efficient bovine insulin presentation capacity of bone marrow-derived macrophages activated by granulocyte-macrophage colony-stimulating factor correlates with a high level of intracellular reducing thiols.

Bone marrow-derived macrophages (BMM phi) were shown before to function as antigen-presenting cells. We show here, that the antigen presentation capacity of BMM phi depends on the nature of the antigen and is differently regulated by the lymphokines interferon-gamma (IFN-gamma) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). When bovine insulin (BI) was employed as antigen, only BMM phi treated with GM-CSF (GM-CSF-M phi) were efficient presenters, but when presentation of the antigens ovalbumin and conalbumin was tested, IFN-gamma-pulsed BMM phi (IFN-gamma-M phi) proved superior to GM-CSF-M phi. The lack of efficient BI presentation function of IFN-gamma-M phi was only obvious, when native BI was used as antigen. Preprocessed BI was presented by IFN-gamma-M phi with drastically higher efficiency than by GM-CSF-M phi. Because processing of insulin depends on reduction of disulfide bonds, we analyzed the content of intracellular reducing thiols within IFN-gamma-M phi, GM-CSF-M phi, and untreated BMM phi. Only after stimulation with GM-CSF did the amount of reduced glutathione and cysteine strongly increase, while IFN-gamma did not efficiently augment the intracellular content of both thiols. These findings suggest that the lymphokines IFN-gamma and GM-CSF differently interfere with the processing capacity of BMM phi by differently regulating the intracellular concentration of the thiols reduced glutathione and cysteine. A high level of these thiols induced by GM-CSF correlates with a prominent capacity to present the antigen bovine insulin.

In vitro effects of monoterpenes on chloroplast membranes.

A chloroplast preparation was extracted from squash (Cucurbita pepo (L.) var. Senator). Enrichment of intact chloroplasts was achieved by continuous free-flow electrophoresis. The addition of monoterpenes, detergent and free fatty acids changed the elecrophoretic separation pattern characteristically. Monoterpene-dependent degradation of envelope membranes could be prevented by addition of α-tocopherol prior to monoterpene incubation.Photosynthetic electron transport of photosystem II was completely inhibited by ß-pinene, Triton X-100 and linolenic acid. Inhibition could be modulated by addition of α-tocopherol or lecithin (phosphatidylcholine) either before or after inhibition by monoterpenes and detergent.Percentage reconstitution of photosynthetic electron transport inhibited by ß-pinene depended on light conditions and incubation time.

Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis of MHC class II molecules and to augmented antigen presentation function.

The effects of granulocyte-macrophage (GM)-CSF on the synthesis of MHC class II molecules and on the Ag presentation capacity by bone marrow derived macrophages (BMM phi) was investigated. BMM phi obtained by in vitro culture in the presence of macrophage-CSF were negative for synthesis of I-A molecules and induced the Ag-mediated proliferation of insulin-specific T clone cells with lower efficiency than splenic accessory cells. After pulse treatment with GM-CSF for 24 to 48 h, day 12 BMM phi exhibited highly efficient Ag presentation function which was superior to that induced by IFN-gamma. Expression of membrane-bound IL-1 was augmented significantly by GM-CSF, but not by IFN-gamma. However, the T cell clone used to probe for accessory cell function of BMM phi was not dependent on IL-1 for optimal proliferation. Concomitantly, GM-CSF induced the de novo synthesis of I-A molecules, although to a lesser extent than optimal doses of IFN-gamma. Thus GM-CSF appears to elicit properties in addition to Ia molecule synthesis and membrane IL-1 expression in BMM phi being essential for efficient accessory cell function to the T clone cells. The activation of BMM phi by GM-CSF was reversible and could be repeated. These data show that GM-CSF exerts a modulatory influence on preformed BMM phi, reversibly activating cells to Ia biosynthetic potential and pronounced accessory cell capacity, thus rendering the explanation unlikely that differentiation of precursor cells into a constitutively functional state had occurred.