In Vitro and In Vivo Interactions of Haemophilus ducreyi with Host Phagocytes

We investigated the phagocytosis of Haemophilus ducreyi both in vitro and in vivo. Human granulocyte and monocyte phagocytosis of opsonized and nonopsonized, fluorescence-labeled H. ducreyi was assessed by flow cytometry. Both Escherichia coli and noncapsulated H. influenzae were included as controls. The maximal percentage of granulocytes taken up by H. ducreyi was 35% after 90 min. In contrast, 95% of H. influenzae bacteria were phagocytosed by granulocytes after 30 min. These results indicated that H. ducreyi phagocytosis was slow and inefficient. Bacterial opsonization by using specific antibodies increased the percentage of granulocytes phagocytosing H. ducreyi from 24 to 49%. The nonphagocytosed bacteria were completely resistant to phagocytosis even when reexposed to granulocytes, indicating that the H. ducreyi culture comprised a mixture of phenotypes. The intracellular survival of H. ducreyi in granulocytes, in monocytes/macrophages, and in a monocyte cell line (THP-1) was quantified after application of gentamicin treatment to kill extracellular bacteria. H. ducreyi survival within phagocytes was poor; approximately 11 and <0.1% of the added bacteria survived intracellularly after 2 and 20 h of incubation, respectively, while no intracellular H. influenzae bacteria were recovered after 2 h of incubation with phagocytes. The role of phagocytes in the development of skin lesions due to H. ducreyi was also studied in vivo. Mice that were depleted of granulocytes and/or monocytes and SCID mice, which lacked T and B cells, were injected intradermally with approximately 10⁶ CFU of H. ducreyi. Within 4 days of inoculation, the granulocyte-depleted mice developed lesions that persisted throughout the experimental period. This result reinforces the importance of granulocytes in the early innate defense against H. ducreyi infection. In conclusion, H. ducreyi is insufficiently phagocytosed to achieve complete eradication of the bacteria. Indeed, H. ducreyi has the ability to survive intracellularly for short periods within phagocytic cells in vitro. Since granulocytes play a major role in the innate defense against H. ducreyi infection in vivo, bacterial resistance to phagocytosis probably plays a crucial role in the pathogenesis of chancroid.     

External quality control assessment in PCR diagnostics of dengue virus infections

BACKGROUND: Increased travelling to countries endemic for dengue fever (DF) demands efficient laboratory diagnostics. Nucleic acid amplification techniques (NAT) are now frequently used for rapid diagnosis of imported viral diseases. Different PCR systems are available. OBJECTIVES: In order to assess the quality of molecular diagnostics of dengue virus infections, an external quality assurance (EQA) in PCR diagnostics was conducted. Study design: A panel of 10 human plasma samples was prepared and spiked with dengue virus types DEN-1 to DEN-4. In addition, a 10-fold dilution series (1:10-1:10(4) ) of DEN-3 virus was included. The panel was pre-tested by nested RT-PCR, in-house real-time PCR, and a commercial real-time PCR kit. The samples were inactivated by gamma irradiation and shipped in freeze dried state. Thirteen laboratories, within the European network for the diagnostics of imported viral diseases (ENIVD) took part using either single-round, nested, or real-time RT-PCR methods. Two laboratories used two methods in parallel, summarising up to 15 comparable results. RESULTS: 33-100% correct results were achieved. All laboratories detected DEN-2 correctly, followed by DEN-1 (14 positive results of 15), DEN-3 (12/15) and DEN-4 (11/15). Testing of the serial dilution revealed low sensitivity in many labs, with results ranging from 33 to 80% of correctly tested samples. CONCLUSION: The EQA gives a feedback of the quality of the RT-PCR system used by each respective laboratory. The different test systems and amplification conditions demonstrate the importance of external quality control measures.     

Accumulation of very long-chain fatty acids does not affect mitochondrial function in adrenoleukodystrophy protein deficiency

X-linked adrenoleukodystrophy (X-ALD, OMIM 300100) is a severe inherited neurodegenerative disease, associated with the accumulation of very long-chain fatty acids (VLCFA). The recent unexpected observation that the accumulation of VLCFA in tissues of the Abcd1-deficient mouse model for X-ALD is not due to a deficiency in VLCFA degradation, led to the hypothesis that mitochondrial abnormalities might contribute to X-ALD pathology. Here, we report that in spite of substantial accumulation of VLCFA in whole muscle homogenates, normal VLCFA levels were detected in mitochondria obtained by organellar fractionation. Polarographic analyses of the respiratory chain as well as enzymatic assays of isolated muscle mitochondria revealed no differences between X-ALD and control mice. Moreover, analysis by electron microscopy, revealed normal size, structure and localization of mitochondria in muscle of both groups. Similar to the results obtained in skeletal muscle, the mitochondrial enzyme activities in brain homogenates of Abcd1-deficient and wild-type animals also did not differ. Finally, studies on mitochondrial oxidative phosphorylation in permeabilized human skin fibroblasts of X-ALD patients and controls revealed no abnormalities. Thus, we conclude that the accumulation of VLCFA per se does not cause mitochondrial abnormalities and vice versa-mitochondrial abnormalities are not responsible for the accumulation of VLCFA in X-ALD mice.     

Beneficial autoimmunity to proinflammatory mediators restrains the consequences of self-destructive immunity

Therapies that neutralize the function of TNF-alpha suppress rheumatoid arthritis (RA) but not osteoarthritis (OA). We show that patients suffering from RA but not OA have significant levels of autoantibodies directed to TNF-alpha. Thus, the immune system can selectively generate autoimmunity to proinflammatory mediators when such a response is beneficial for the host. A well-defined model of RA was used to elaborate the contribution of beneficial autoimmunity to the regulation of disease. We show that during the disease autoantibody production is elicited against few inflammatory, but not regulatory, mediators. Selective amplification of these beneficial antibodies by targeted DNA vaccines provided protective immunity. Epitope mapping revealed that anti-TNF-alpha immunity is highly restricted and excretes no crossreactivity to other known gene products. Its selective exclusion substantially exacerbated the disease. Administration of anti-TNF-alpha antibodies could then override this aggravation. This substantiates the significance of beneficial autoimmunity in restraining self-destructive immunity.     

Prolongation of small bowel allograft survival with a sequential therapy consisting of a synthetic MHC class II peptide and temporarily low-dose cyclosporine A

It has been extensively documented the role of the indirect pathway of allorecognition in allograft rejection. However, recent data demonstrate that the manipulation of this pathway could be also sufficient to promote prolongation of allograft survival. In the present study we evaluated the effect of preoperative immunization with the WF-specific MHC class II peptides RT1.D2 and RT1.B2 in combination with low-dose CsA from days 0 to 7 (5 mg/kg/day) and from days 8 to 30 (1 mg/kg/day) after WF small bowel transplantation. Seven days before and on the day of transplantation, LEW recipients were immunized with the two WF MHC class II peptides RT1.B2 and RT1.D2. The CsA monotherapy induced an allograft survival of 49.3 +/- 6.1 days. MHC class II peptide immunization had a limited effect on allograft survival for RT1.D2 (47.1 +/- 3.8 days) and induced prolongation of allograft survival for RT1.B2 (73.6 +/- 34.6 days). This effect seems to be based on the absence or silence of RT1.B2-reactive T cells and rejection seems to be correlated with the presence of RT1.B2-specific T cells in the late phase. Therefore, the combination of RT1.B2 with low-dose CsA shifts the immunological response and protects small bowel allograft rejection.     

Knowledge about asthma and COPD: associations with sick leave, health complaints, functional limitations, adaptation, and perceived control

We sought to investigate associations between knowledge about the disease and sick leave, health complaints, functional limitations, adaptation and perceived control. Patients with asthma (n = 101) and COPD (n = 64) underwent lung function tests and completed questionnaires. In addition, all were asked the question: ‘what is the diagnosis of your disease?’, with the response categories: ‘asthma’ and ‘COPD (chronic bronchitis or emphysema)’. Thirty-five percent of the asthma patients and 30% of the COPD patients did not know their correct diagnosis. Sick leave was not associated with knowledge about the disease in asthma and COPD. In asthma, much knowledge about management of the disease was associated with better adaptation (P = 0.01) and less perceived control over health by external factors (P = 0.02). Knowing the correct diagnosis was associated with less control over health by powerful others (P = 0.02). For COPD, more knowledge about management of the disease was associated with better adaptation (P = 0.02) and less control over health by internal factors (P = 0.01). Knowing the correct diagnosis was associated with less control over dyspnea at work (P = 0.01).     

Decreased phenylalanine uptake and turnover in patients with vitiligo

The human epidermis has the full machinery for autocrine L-phenylalanine turnover to L-tyrosine in keratinocytes and melanocytes. Phenylalanine hydroxylase (PAH) activities increase linearly with inherited skin colour (skin phototype I-VI, Fitzpatrick classification) yielding eightfold more activities in black skin compared to white skin. Moreover, UVB irradiation (1 MED) significantly increases epidermal PAH activities 24 h after exposure. Importantly, L-phenylalanine uptake and turnover in the pigment forming melanocytes is vital for initiation of melanogenesis. In this context it was shown that the uptake of this amino acid is regulated by calcium. The depigmentation disorder vitiligo provides a unique model to follow impaired L-phenylalanine turnover in the skin as well as in serum because affected individuals hold an impaired epidermal 6BH4 de novo synthesis/recycling and regulation including low epidermal PAH activities. After overnight fasting and oral loading with L-phenylalanine (100 mg/kg body weight), 29.6% of 970 patients tested (n=287/970) yielded serum phenylalanine/tyrosine ratios >or=4 and 35.3% (n=342/970) had mild to moderate hyperphenylalaninaemia (HPA), while 9.3% (n=90/970) had both serum L-phenylalanine levels >or=2.0 mg/dl and phe/tyr ratios >or=4.0. Isolated HPA was found in 26% (n=252/970), whereas 20.3% had only increased ratios (n=197/970). None of the patients had phenylketonuria and the family history for this metabolic disease was negative. The IQ followed normal Gaussian distribution. In vitro L-phenylalanine uptake/turnover studies on primary epidermal melanocytes originating from these patients demonstrated a significantly decreased calcium dependent L-phenylalanine uptake and turnover compared to healthy control cells. Based on our observation, we would like to propose that phenylalanine uptake/turnover is under tight control by calcium which in turn could offer an additional novel mechanism in the aetiology of HPA.     

Renin gene and angiotensin II AT1 receptor gene expression in the kidneys of normal and of two-kidney/one-clip rats

This study aimed to investigate the inter-relation between the angiotensin II (ANG II) AT₁ receptor and renin gene expression in rat kidneys. To this end, renin mRNA levels and mRNA levels for AT_1a and AT_1b were assayed by RNase protection in the kidneys of normal rats, in animals treated with the AT₁ antagonist losartan and in rats bearing 0.2-mm left renal artery clips for 2 days. In normal rats, we found a negative correlation between renin mRNA levels and AT_1a receptor mRNA levels. Losartan led to a fourfold increase in renin mRNA levels without changing AT₁ receptor mRNA levels. Unilateral renal artery clipping increased renin mRNA levels fourfold in the clipped kidney and suppressed renin mRNA levels in the contralateral kidneys. AT₁ receptor mRNA levels were not changed in the contralateral intact kidneys, but were significantly decreased by 15–25% in the clipped kidneys. Renin mRNA levels were inversely correlated to AT_1a mRNA levels in the clipped, but not in the contralateral, kidneys. Our findings suggest that the systemic activity of the renin angiotensin system has no regulatory influence on renal AT₁ receptor gene expression. Renin mRNA levels in normal and in clipped kidneys appear to be negatively determined by the level of AT_1a receptor gene expression. Thus modulation of AT_1a receptor gene expression could be a pathway for indirect modulation of renin gene expression by ANG II. This conclusion is in agreement with the observation that AT₁ receptor antagonists are powerful stimulators of the renin system.