Insufficient tetanus vaccination status in patients with chronic leg ulcers. Results of a prospective investigation in 100 patients.

BACKGROUND: Tetanus disease is caused by Clostridium tetani and is one of the most common infectious diseases worldwide. Despite international recommendations for patients with a chronic leg ulcer, there has been a distinctive lack of protection provided by vaccination for these patients in the past decades. METHODS: Within the context of our prospective clinical investigation we consecutively determined the concentrations of immunoglobulin G antibodies against C. tetani in 100 patients with a chronic leg ulcer between January 2005 and November 2006. RESULTS: A total of 38 patients were male, and 62 were female. Their mean age was 71 years (25-94). In a total of 47% (n = 47; 13 male, 34 female, mean age: 76 years) of the patients, insufficient immunoglobulin G antibody concentrations were detected. Particularly the subanalysis indicated an insufficient tetanus protection provided by vaccination in 70% of the people aged >or=80 years. CONCLUSION: A chronic wound, e.g. in the form of a leg ulcer, is known as a potential entrance for C. tetani. Unlike acute wounds, however, it is hardly ever considered to be a reason for assessment of the tetanus immune status. The results of our investigation clarify that particularly elderly people suffering from a leg ulcer have to be tested for tetanus protection provided by vaccination more strictly than ever, and if necessary, vaccinations have to be renewed.

Behcet's disease is associated with increased concentrations of antibodies against phosphatidylserine and ribosomal phosphoproteins.

BACKGROUND: Although Behcet's disease (BD) is classified among the vasculitides laboratory diagnostic does not include regularly autoantibodies associated with vascular manifestations of systemic autoimmune diseases. PATIENTS AND METHODS: Twelve consecutive BD patients were studied for autoantibodies associated with vascular manifestations of systemic autoimmune diseases, HLA frequencies, and possible neurological involvement using neurophysiological methods and MRI. RESULTS: HLA-C*15 and C*16 frequencies were significantly (p < 0.05) higher in the patients compared with a reference population. Immunoglobulin G concentrations of antiphosphatidylserine and antiribosomal phosphoprotein antibodies were significantly elevated in BD patients when compared with healthy controls. CONCLUSIONS: The increased frequencies of HLA-C alleles in BD patients may stress the role of NK cells in the pathogenesis of this disease. Antiphosphatidylserine autoantibodies may in view of their role in apoptosis be involved in the development of vasculitis in BD. Because concentrations of antiphosphatidylserine and antiribosomal phosphoprotein antibodies were increased in BD diagnostic tools of this disease should be extended with humoral parameters associated with vascular manifestations of systemic autoimmune diseases.

Immunosuppression with FK506 increases bone induction in demineralized isogeneic and xenogeneic bone matrix in the rat.

The aim of the present study was to investigate a systemic induction of bone formation in rats by immunosuppression with FK506 (1 mg/kg body weight intraperitoneally [ip]) in a model of osteoinduction of isogeneic and xenogeneic demineralized bone matrix (DBM) for a period of 28 days. In particular, alterations of in vitro cytokine synthesis and changes of lymphocyte subsets were studied. DBM was implanted intramuscularly in the abdominal wall of Lewis rats (seven per group). Blood was sampled on days -7, 0, 7, and 28 for determination of in vitro tumor necrosis factor a (TNF-alpha) synthesis and lymphocyte subsets by flow cytometry (CD3+, CD4+, CD8+, CD45+, ED9+, and Ia+ antibodies). Ossicles of de novo formed bone and the tibias were removed on day 28 after double tetracycline labeling for histomorphometric analysis. Immunosuppression with FK506 significantly decreased lipopolysaccharide (LPS)-stimulated in vitro cytokine synthesis after 7 days and 28 days (p < 0.05). Compared with control animals FK506 treatment significantly increased the volume of induced bone in isogeneic (2.1 +/- 0.3 mm3 vs. 10.8 +/- 0.9 mm3) and xenogeneic (O mm3 vs. 4.7 +/- 0.8 mm3) DBM. Bone histomorphometry of the tibias revealed that immunosuppression increased both bone formation and bone resorption, accompanied by a significant reduction in the relative trabecular area (Tb.Ar). FK506 caused a decrease in the counts of CD8+ T cells probably because of destruction or dislocation of these cells. This suggests that the amount of CD8+ cells and the degree of T cell activation in terms of mean fluorescence intensity (MFI) may be associated with bone metabolism. In support of this, statistical analysis revealed a significant positive correlation between parameters of bone formation as well as bone resorption and the CD4+/CD8+ ratio. There was a significant negative correlation between parameters of remodeling of the metaphysis of the tibia and induced bone volume (BV), respectively, and MFI values of CD3+/Ia+ cells. These findings suggest an important role of T lymphocytes in bone formation and bone resorption in vivo. FK506 caused a marked increase of bone formation in DBM. However, the conclusion that immunosuppression increases fracture healing warrants further investigation.

Cimetidine and the immuno-response in healthy volunteers.

We compared the immunologic measurements from treatment of 12 healthy volunteers (six male, six female) with 800 and 1,600 mg cimetidine. In the first trial 800 mg cimetidine was administered daily to the volunteers over a period of 7 d; after an interruption of 2 months, 1,600 mg of cimetidine was applied daily for 21 d. The most striking result of our study was an increased mitogen-induced lymphocyte proliferation. This conclusion can be drawn from the fact that phytohaemagglutinin (PHA) (0.4 microgram/well) and pokeweed mitogen (PWM) (0.4 microgram/well) induced lymphocyte proliferation were found to be significantly increased in comparison to pretreatment values on day 7 in both cimetidine regimens (800 mg; PHA: mean proliferation 66,500 before treatment to 166,00 cpm, PWM: mean proliferation 8,800 before treatment to 34,000 cpm; 1,600 mg; PHA; mean proliferation 48,700 before treatment to 81,600 cpm; PWM: mean proliferation 6,300 before treatment to 16,200 cpm). Increased mitogen-induced proliferation following cimetidine intake is of special interest because the mechanisms of this activation process are incompletely known. Lymphocyte proliferation response is dependent on the availability of extracellular calcium. The function of the other bivalent cations is unknown. We found that the extent of mitogen-induced lymphocyte proliferation correlates with cellular intralymphocytic zinc and magnesium amounts (coefficients of correlation [r]) (800 mg: PHA/Mg r = 0.84; PHA/Zn r = 0.86; PWM/Mg r = 0.88; PWM/Zn r = 0.87). Though the application of both cimetidine doses causes enhanced mitogen-induced lymphocyte proliferation on day 7, T lymphocytes with different phenotypic properties appear to be influenced by cimetidine. In the first dose regimen (800 mg) the number of the CD8 lymphocytes decreased significantly from 16.1% (365 cell/microliters blood) to 12.7% (264 cells/microliters blood) after 7 d of cimetidine intake. After the period of high-dose (1,600 mg) cimetidine administration (at day 21) numbers of CD4 lymphocytes were significantly increased from 41.5% (860 cells/microliters blood) to 56.3% (1,210 cells/microliters blood). Our results show that although different cimetidine doses obviously influence different cell types of healthy volunteers, the cellular mechanisms are the same, namely, a proliferation and an increased incorporation of magnesium and zinc in lymphocytes.

The G protein beta3 subunit 825T allele is a genetic marker for enhanced T cell response.

The G protein beta3 subunit (GNB3) 825T allele is predictive of enhanced Gi protein activation. Studying the influence of C825T allele status on cellular in vitro immune responses towards recall antigens and interleukin-2 stimulation we observed a 2-4-fold, significantly increased proliferation in homozygous 825T (TT) vs. C825 allele (CC) carriers. Furthermore, lymphocyte chemotaxis and CD4(+) T cell counts of individuals with TT+TC genotypes were significantly enhanced compared to the CC genotype. In summary, it appears that C825T allele status is highly predictive of immunocompetence and could be a candidate gene in disorders associated with inadequate immune response.

Relative and absolute numbers of human lymphocyte subpopulations. A comparison of immunofluorescence microscopy and flow cytometric methodologies with special reference to precision and reference values.

Lymphocyte subpopulations were determined in blood samples from blood donors (40 women and 45 men) using immunofluorescence microscopy and flow cytometric methodologies. The study demonstrates the value of both methods for the enumeration of lymphocyte subpopulations. The advantages of employing an automated flow cytometer system are better precision and speed. The automated systems require a large initial technical and financial burden and are therefore probably destined to be reserved for the larger laboratory. There is a need for an adequate lymphocyte standard which shows little variation between aliquots and can be used for interlaboratory comparisons.

Impact of localized radiotherapy on blood immune cells counts and function in humans.

Immune cells subsets were prospectively analyzed after localized radiotherapy (LRT). LRT reduced the levels of all lymphocyte subsets, with B-cells and naive T-cells being most sensitive. Lymphocyte function was suppressed, but still within the normal range. Rapid recovery of cytotoxic T-cells/natural killer cells after LRT and the functional suppression within normal levels explains the low incidence of infections after LRT.

Influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) on whole blood endotoxin responsiveness following trauma, cardiopulmonary bypass, and severe sepsis.

Major surgery, multiple injury, and severe sepsis lead to an impaired immune response. The suppressed status of the immune system is reflected by a reduced TNFalpha production of whole blood after stimulation with endotoxin in vitro and by a decreased HLA-DR expression on monocytes. In the present study, the effect of the immunostimulating hematopoetic growth factor GM-CSF on whole blood cultures of multiple injury, cardiac surgery, and severe sepsis patients was investigated. Endotoxin-induced TNFalpha production and HLA-DR expression was reduced in blood cultures of these patients compared to healthy donors. Preincubation with GM-CSF in vitro increased cytokine production in volunteers' and all patients' blood specimens in a dose-dependent manner. The elevation of cytokine response in cardiopulmonary bypass patients' blood, caused by in vitro preincubation with GM-CSF, equaled that of normal patients, whereas GM-CSF caused a lower rise of TNFalpha-producing capacity in blood of multiple-injury and sepsis patients. Further, GM-CSF treatment in vitro increased the down-regulated HLA-DR expression on monocytes prepared after cardiac surgery to a degree comparable to preoperative levels. Finally, GM-CSF incubation in vitro elevated TNFalpha synthesis in normal monocytes and in cells treated with a combination of the anti-inflammatory mediators IL-10, TGFbeta, and PGE2. These experiments show that hyporesponsiveness of whole blood induced by trauma, sepsis, or cardiac surgery is not irreversible but can be, at least in vitro, overridden by the immunostimulating compound GM-CSF.

Association of increased bronchoalveolar lavage fluid albumin and serum beta 2-microglobulin with pulmonary complications after allogeneic bone marrow transplantation.

The aim of this prospective study was to identify markers in bronchoalveolar lavage fluid (BAL fluid) and serum predictive for the development of pulmonary complications in the early phase (< 50 days) post-BMT. Concentrations of BAL fluid albumin (alb) and serum beta 2-microglobulin (S-beta 2m,) were determined 10 days before BMT (BAL-B, baseline) and on day 1 post-BMT (BAL-1) in 20 patients who subsequently developed pulmonary complications (group 1) and in 66 patients who remained free of complications for a minimum of 12 months (group 2). Median BAL fluid alb concentrations were significantly (P < 0.05) higher in group 1 patients as compared to group 2 patients at BAL-B (40 vs 28 mg/l) and at BAL-1 (30 vs 15 mg/l). S-beta 2m at BAL-1 was also significantly elevated in group 1 patients (median 1.3 mg/l) compared to group 2 patients (median 1.15 mg/l). Using cut-off values for BAL fluid alb (> 23 mg/l) and S-beta 2m (> 0.8 mg/l) we identified 12 patients out of 19 who developed subsequent pulmonary complications from 12 out of 62 patients without such complications, 1 day post-BMT.

Effect of atorvastatin and clopidogrel on cellular immune function.

Lipid lowering therapy by statins and antiaggregation have become the basis of any anti-atherosclerotic prophylaxis either as primary or secondary prophylaxis. As several recent papers indicated immunosuppressive properties of statins we investigated changes in lymphocyte subpopulations, apoptosis markers, and cellular immune response towards mitogens after a short-term therapy with atorvastatin and clopidogrel. Nine healthy volunteers (four male, five female, age ranging from 26 to 43 years) were treated with 20 mg atorvastatin for 4 weeks and for 2 additional weeks with 20 mg atorvastatin and 75 mg clopidogrel after oral consent was given. Lymphocyte subpopulations were counted by flow cytometry. To assess cellular in vitro immune function, lymphocyte transformation tests with four mitogens (PHA, ConA, PWM, and OKT3) were performed. Absolute leucocyte counts remained unchanged as well as the granulocyte, monocyte, lymphocyte, and lymphocyte subpopulation counts. There were no detectable changes in markers of cell activation (HLA-DR, CD25, CD69, and CD86) or apoptosis (CD95, annexin). Cellular in vitro responses towards four mitogens did not show significant changes after atorvastatin nor after atorvastatin plus clopidogrel treatment.In conclusion, our data show that atorvastatin is not an immunosuppressive drug under therapeutical conditions.

Coincidence of normal tension glaucoma, progressive sensorineural hearing loss, and elevated antiphosphatidylserine antibodies.

BACKGROUND: Recently, in patients with normal tension glaucoma (NTG) elevated levels of antiphosphatidylserine antibodies (APSA), a subgroup of antiphospholipid antibodies (APLA) were found. Progressive sensorineural hearing loss (PSHL) is associated with autoimmune diseases and also the presence of APLA. METHODS: To investigate a possible association between NTG and PSHL, 34 patients (age range 31-81 years) with NTG were evaluated for evidence of audiovestibular disorders. Besides ophthalmological standard examinations (slit lamp, IOP, funduscopy, perimetry) scanning laser tomography and polarimetry were performed. From all patients' audiograms, stapedial thresholds and otoacoustic emissions were obtained. The serological testing of patients and controls (40 healthy blood donors older than 50 years) concerned IgG and IgM levels of antibodies against phosphatidylserine (APSA) and beta2 glycoprotein. RESULTS: 23 of 34 NTG patients had hearing loss (PSHL n = 11; presbyacusis n = 12). The NTG patients had significantly higher APSA levels than controls. Elevated APSA concentrations were significantly more frequent in patients with NTG and hearing loss compared with NTG patients with normacusis. CONCLUSIONS: These findings show that NTG and hearing loss have a high coincidence. The elevated APSA levels may indicate an association with similar systemic autoimmune processes.

Nodular pulmonary vasculitis in a twelve-year-old boy.

A 12-year-old boy presented with left shoulder pain during physical exercise and complained of uncommon sweating and fatigue. Diagnostic evaluation revealed a solitary pulmonary nodule in the left upper lobe. All laboratory values were within normal limits, except for an elevated level of antineutrophil cytoplasmic antibodies directed against myeloperoxidase (p-ANCA). Surgery was performed, and pathological examination showed a localized granulomatous vasculitis. Antineutrophil cytoplasmic antibodies directed against affinity purified proteinase 3 (p-ANCA) concentrations returned to baseline within 6 months, and the patient has done well during a follow-up period of 2 years. While nodular vasculitis is known to occur in Wegener's granulomatosis, to the best of our knowledge, this case represents the first c-ANCA negative primary pulmonary vasculitis in childhood.

Antagonism of the estradiol-mediated repression of microsomal 3 beta-hydroxysteroid dehydrogenase activity in rat liver by antiestrogenic substances.

5 alpha-Dihydrotestosterone, 17-hydroxyprogesterone caproate, 2-methoxyestrone and a number of nonsteroidal antiestrogens (clomiphene citrate, nafoxidine hydrochloride, tamoxifen, MER-25) were tested for their ability to block estradiol-mediated repression of the androgen-dependent 3 beta-hydroxysteroid dehydrogenase activity of male rat liver. With the exception of 5 alpha-dihydrotestosterone, which induced activity in females, none of these substances affected 3 beta-hydroxysteroid dehydrogenase activity when administered alone to otherwise untreated male and female rats. Tamoxifen (100 or 500 micrograms/day) was the only substance which prevented a decrease in enzyme activity when given simultaneously with estradiol (5 micrograms/day). The estradiol-mediated decrease in activity was not antagonized by a 100-fold higher dose of androgen (5 alpha-dihydrotestosterone, 0.5 mg/day), demonstrating the potent antiandrogenic effect of estradiol on this hepatic androgen-dependent enzyme activity.

Thymidine utilization abnormality in proliferating lymphocytes and hepatocytes of the woodchuck.

Effective incorporation of tritiated thymidine ([(3)H]TdR) into proliferating lymphocytes is important because [(3)H]TdR is a standard label to study proliferate T-cell responses. We analyzed the thymidine utilization of woodchuck peripheral blood lymphocytes (PBL) since the [(3)H]TdR incorporation assay was not applicable to measure proliferative immune responses in the woodchuck, a current major virus/host model for human hepatitis B virus infection. Incorporation of [(3)H]TdR into DNA as well as the activity of the salvage pathway enzyme thymidine kinase (TK) of proliferating woodchuck PBL was low compared to human lymphocytes. Furthermore, [(3)H]TdR incorporation of proliferating woodchuck PBL remained residual regardless of the use of methotrexate, an inhibitor of the competitive deoxythymidine monophosphate de novo synthesis pathway. Using a human probe, specific for the proliferation-associated TK1, we proved the genomic presence and transcription of TK1 sequences in various species. TK1 sequences were detected in the genome of human, mouse, woodchuck, and chicken specimens. In contrast to proliferating human PBL and 3T3 mouse fibroblasts, no TK1 transcript was found in proliferating woodchuck PBL and hepatic cells. Transfection experiments with vectors containing the murine or human TK1 and selection assays demonstrated the ability of woodchuck cells to transcribe TK1 and to express functional TK1 proteins. Our study characterizes the unique failure of sufficient [(3)H]TdR incorporation into proliferating woodchuck cells and demonstrates tritiated adenine and serine as alternative labels to monitor PBL proliferation in the woodchuck.

Relevance of low titers of cryoglobulins and cold-agglutinins in patients with isolated Raynaud phenomenon.

INTRODUCTION: A Raynaud phenomenon can be associated with cold agglutinins or cryoglobulins. Although cold agglutinins or cryoglobulins may lead to severe acral gangrene the finding of relevant titers is rare. Low titers of cold agglutinins or cryoglobulins are detected more frequently but are assumed to be without any importance. OBJECTIVES: To prove a possible diagnostic or prognostic role of low titers of cold agglutinins or cryoglobulins in patients presenting an isolated Raynaud phenomenon we did a retrospective analysis. SETTINGS AND SUBJECTS: In 306 patients (40+/-16 years, range: 15-83 years) with a mean duration of the history of an isolated Raynaud phenomenon of 48+/-73 months we did a clinical examination, an analysis of antinuclear antibodies, extractable antibodies, cold agglutinins, cryoglobulins, plasma and blood viscosity, erythrocyte aggregation and a nail fold capillaroscopy. RESULTS: Low titers of cold agglutinins were found in 49 patients and of cryoglobulins in 7 patients. The finding of such low titers was not associated with extensive clinical symptoms, duration of clinical symptoms, megacapillaries or haemorrhagies in capillaroscopy, pathologic plasma and blood viscosity and erythrocyte aggregation. The follow-up investigations (mean: 3.1+/-1.2 years, range: 3-7 years) revealed no development of a haematological, vasculitic or connective tissue disease in the subgroup of patients who only had low titers of cold agglutinins. CONCLUSION: The detection of low titers of cold agglutinins in patients with isolated Raynaud phenomenon is of no diagnostic or prognostic relevance.

Thrombangitis obliterans: leucocyte subpopulations and circulating immune complexes.

BACKGROUND: The etiology of thrombangitis obliterans is still unclear. Although cellular infiltration of the vessel wall is known, no studies on peripheral blood mononuclear cells are reported. Therefore, we assessed leucocyte subpopulations and circulating immune complexes in patients with thrombangitis obliterans and a control group of normal people. PATIENTS AND METHODS: 31 patients (40 +/- 2 years, 24 male, 7 female) with thrombangitis obliterans were included, based on the following criteria: age of manifestation, acral ischemia in legs and arms, previous thrombophlebitis or phlebitis saltans. Manifestation of atherosclerosis or other vasculitic manifestations were excluded. Leucocyte subpopulations, levels of C-reactive protein (CRP) and circulating immune complexes (CIC) were investigated. An age-matched control group (n = 25) was recruited from voluntary blood donors. RESULTS: Leucocyte counts in the thrombagitis group (mean +/- SD: 10,839 +/- 782/nl) were significantly different from the control group (6205 +/- 414/nl, p < 0.0001). The same was true for absolute counts of granulocytes, monocytes and lymphocytes. The results were independent from CRP, which was elevated only in 6 patients. Relative counts of naive helper T-cells were significantly lower in the patient group. HLA-DR expression on B-cells was lower on the patients' lymphocytes. The concentrations of IgA, IgG and IgM in CIC were higher in the thrombangitis patients compared to the control group. C1q-binding capacity and phosphatidylserine antibodies showed no differences. CONCLUSIONS: Patients suffering from thrombangitis obliterans show alterations of leucocyte counts and their subpopulations as well as alterations of the humoral (IgCIC) immune system.