Rats and mice immunised with chimeric human/mouse proteinase 3 produce autoantibodies to mouse Pr3 and rat granulocytes

Aim

In this study, we employed chimeric human/mouse Proteinase 3 (PR3) proteins as tools to induce an autoantibody response to PR3 in rats and mice.

Method

Rats and mice were immunised with recombinant human PR3 (HPR3), recombinant murine PR3 (mPR3), single chimeric human/mouse PR3 (HHm, HmH, mHH, mmH, mHm, Hmm) or pools of chimeric proteins. Antibodies to mPR3 and HPR3 were measured by ELISA. Antibodies to rat PR3 were determined by indirect immunofluorescence (IIF) on rat white blood cells. Urinalysis was performed by dipstick analysis. Kidney and lung tissue was obtained for pathological examination.

Results

In mice, immunisation with the chimeric human/mouse PR3 Hmm led to an autoantibody response to mPR3. Rats immunised with the chimeric human/mouse PR3 Hmm, HmH and mmH, or a pool of the chimeric human/mouse PR3 proteins, produced antibodies selectively binding to rat granulocytes as detected by IIF. No gross pathological abnormalities could be detected in kidney or lungs of mice or rats immunised with chimeric human/mouse PR3.

Conclusion

Immunisation with chimeric human/mouse proteins induces autoantibodies to PR3 in rats and mice. Chimeric proteins can be instrumental in developing experimental models for autoimmune diseases.Wegener''s granulomatosis (WG) is associated with antineutrophil cytoplasmic antibodies (ANCA),¹ in particular to Proteinase 3 (PR3).² PR3‐ANCA are a specific and sensitive marker for WG, whereas other ANCA‐associated vasculitides are associated with antimyeloperoxidase (MPO) antibodies.³ The association between fluctuations in PR3‐ANCA and relapsing disease in WG suggests a pathogenic role for PR3‐ANCA.⁴ However, although animal models support a pathogenic role for MPO‐ANCA in vasculitis development⁵ attempts to develop an animal model for PR3‐ANCA‐associated vasculitis have not been successful thus far.⁶ Recently, chimeric Pr3 proteins that are partly composed of the human amino acid sequence and partly of the sequence of the mouse homologue have been described.⁷ In this study, we employed these chimeric proteins as immunological tools to induce an autoantibody response in rats and mice to PR3. We hypothesised that, by epitope spreading,⁸ antibodies could develop to rat or mouse PR3 leading to an autoimmune response to PR3. As the homology between rat and mouse PR3 is 94%, antibodies to mouse PR3 will likely also recognise rat PR3.⁹In a set of experiments, rats and mice were immunised with separate chimeric human/mouse PR3 proteins or combinations thereof. Indeed, rats immunised with chimeric human/mouse PR3 developed autoantibodies to mouse PR3 and rat granulocytes and mice immunised with one specific chimeric human/mouse PR3 induced antibodies to mouse PR3. The results provide the first evidence that an autoantibody response can be generated in rats and mice by immunisation with chimeric proteins.     

A comparison of hexaminolevulinate fluorescence cystoscopy and white light cystoscopy for the detection of carcinoma in situ in patients with bladder cancer: a phase III, multicenter study

PURPOSE: We compared hexaminolevulinate (Hexvix) fluorescence cystoscopy with white light cystoscopy for detecting carcinoma in situ. MATERIALS AND METHODS: In this multicenter study 298 patients with known or suspected bladder cancer underwent bladder instillation with 50 ml 8 mM hexaminolevulinate for 1 hour. Cystoscopy was then performed, first using standard white light and then hexaminolevulinate fluorescence cystoscopy. Lesions or suspicious areas identified under the 2 illumination conditions were mapped and biopsied for histological examination. In addition, 1 directed biopsy was obtained from an area appearing to be normal. RESULTS: Of 196 evaluable patients 29.6% (58 of 196) had carcinoma in situ, including 18 with carcinoma in situ alone, and 35 with carcinoma in situ and concomitant papillary disease, which was only detected on random biopsy in 5. Of the 18 patients with no concomitant papillary disease carcinoma in situ was detected only by hexaminolevulinate fluorescence in 4 and only by white light in 4. In the group with concomitant papillary disease carcinoma in situ was found only by hexaminolevulinate fluorescence in 5 patients and only by white light in 3. The proportion of patients in whom 1 or more carcinoma in situ lesions were found only by hexaminolevulinate cystoscopy was greater than the hypothesized 5% (p=0.0022). Overall more carcinoma in situ lesions were found by hexaminolevulinate than by white light cystoscopy in 22 of 58 patients (41.5%), while the converse occurred in 8 of 58 (15.1%). Biopsy results confirmed cystoscopy findings. Of a total of 113 carcinoma in situ lesions in 58 patients 104 (92%) were detected by hexaminolevulinate cystoscopy and 77 (68%) were detected by white light cystoscopy, while 5 were detected only on directed visually normal mucosal biopsy. Hexaminolevulinate instillation was well tolerated with no local or systemic side effects. CONCLUSIONS: In patients with bladder cancer hexaminolevulinate fluorescence cystoscopy with blue light can diagnose carcinoma in situ that may be missed with white light cystoscopy. Hexaminolevulinate fluorescence cystoscopy can be used in conjunction with white light cystoscopy to aid in the diagnosis of this form of bladder cancer.     

Follow-up study of blindness attributed to cataract in Karnataka State,India

AIM. Presentation of the results of rapid assessments of bilateral cataract blindness in persons 50 years of age and older in 19 districts of Karnataka State, India. MATERIALS. A total of 21,950 persons 50 years of age and older in 19 out of 20 districts were examined. In each district, 15 clusters were randomly selected and in each cluster the visual acuity and lens status were assessed in 90 persons 50 years of age and older. METHODS. Systematic Random Cluster Sampling was used. Assuming a prevalence of at least 4.3% and a design effect of 1.5, the survey was designed to give an estimated prevalence with a sampling error of 20% or less at 80% confidence. Visual acuity was measured with a tumbling E chart at 6 meters distance with available correction. Lens status was assessed by distant direct ophthalmoscopy with undilated pupil under semi-dark conditions. RESULTS. The average age and sex adjusted prevalence of cataract blindness was 4.93%, with a variation of 1.58% to 7.24% in different districts. The prevalence in females was higher than in males. Cataract Surgical Coverage, an indicator for coverage and service utilization, varied from 42% to 68% in different districts. On average, males had a higher coverage than females. Of all aphakic eyes in the sample, 26.4% could not see 6/60. Barriers to cataract surgery are linked to service providers. CONCLUSIONS. Rapid assessments for cataract blindness in persons aged 50 years and older can be conducted at district level in India with existing resources and at affordable costs. The results suggest an increase in cataract blindness since the previous survey of 1986. The long-term visual outcome needs improvement. Change in barriers to cataract surgery requires a shift in health education strategy and messages. The large variation in prevalence justifies district-level surveys.     

Impact of stroke type on survival and functional health.

In a cohort 760 consecutive stroke patients (23 hospitals in the Netherlands), we studied prognosis in relation to stroke type and focused on (a) short-term and long-term mortality, and (b) long-term functional health. Based on clinical and CT data, we distinguished infratentorial strokes from supratentorial strokes (lacunar infarctions, (sub)cortical infarctions and intracerebral hemorrhages). Cumulative mortality for all stroke patients was 34% at 6 months, 51% at 3 years, and 62% at 5 years. Short-term mortality could be explained by stroke type, whereas long-term mortality could not. Of all survivors, 55% were in poor functional health at 6 months, 49% at 3 years and 42% at 5 years. Long-term functional health outcomes were associated with stroke type. We conclude that the impact of stroke type on mortality is limited to the first 6 months, whereas the type of stroke influences the long-term functional health.     

Dorsal lunate tilt (DISI configuration): sign of scaphoid fracture displacement

Excessive dorsiflexion (dorsal tilting) of the lunate on a lateral wrist radiograph can be an important sign of carpal injury. Lunate dorsiflexion is a well-recognized sign of an intercarpal ligamentous injury pattern known as dorsal intercalated segment instability (DISI). It is less well recognized that excessive dorsal tilting of the lunate (DISI configuration) can also be produced by displacement of a scaphoid waist fracture. Since the management and prognosis of displaced scaphoid fractures may be quite different from those for nondisplaced fractures, radiologists can make an important contribution by recognizing dorsal tilting of the lunate and appreciating that it may be an important, indirect sign of scaphoid fracture displacement, which may not be directly visualized with standard wrist radiography. In this setting, computed tomography or complex motion tomography may be helpful for further evaluation of the scaphoid fracture.     

Inflammatory clearance of apoptotic cells after UVB challenge

In the human body, every day billions of apoptotic cells are produced. Removal of these cells is necessary, to prevent the release of intracellular toxic constituents, and occurs very effectively via phagocytosis by (semi)-professional phagocytes. This elimination process occurs rapidly and without inflammation. In systemic lupus erythematosus (SLE) a disturbed elimination of apoptotic cells has been implicated in the induction and reactivation of the disease. Accumulation of apoptotic cells may result in autoantibody formation. A delayed, pro-inflammatory clearance is also thought to play a crucial role in the development of inflammatory lesions once the disease has manifested. One of the hallmarks of patients with SLE is the development of cutaneous lesions upon exposure to sunlight. In this review, we will focus on apoptotic cells, their elimination, and the consequences of a disturbed elimination of apoptotic cells on the development of UVB induced inflammatory skin lesions.     

Obesity: Risk Factor for Increased Resource Utilization at Bidirectional Glenn

Background

Underweight infants with single‐ventricle cardiac physiology have been shown to have increased morbidity, mortality, and resource utilization. The purpose of this study was to determine whether patients who were overweight, as defined by weight‐for‐length z score >90th percentile, were similarly at risk for increased resource utilization, as defined by mechanical ventilation hours (VHs) and intensive care unit length of stay (ICU LOS).

Methods

We evaluated resource utilization for 109 patients from our institution who underwent bidirectional Glenn surgery from January 2010 to June 2015 and met prespecified inclusion criteria. Patients were divided into 3 groups: underweight (z score, <5th percentile), normal weight (z score, 5th–90th percentile), and overweight (z score, >90th percentile).

Results

ICU LOS was longer in the overweight group (median, 18.5 days) when compared with the under‐ and normal‐weight groups (median LOS, 11 and 9 days, respectively) but did not reach statistical significance. VHs were also increased in the overweight group (median, 72 hours) when compared with the underweight (median, 27 hours) and normal weight (median, 25 hours) groups. This increase in VHs was statistically significant (P = .03).

Conclusions

This study suggests that patients with single‐ventricle physiology who are overweight at the time of their bidirectional Glenn surgery may be at risk for increased resource utilization as compared with those who meet or fail to meet their caloric recommendations. These findings represent an underappreciated risk factor in this already‐vulnerable patient population, providing potential opportunity for intervention and improved outcomes.