Human neutrophil dysfunction with giant granules and defective activation of the respiratory burst

We describe a patient whose peripheral blood neutrophils and bone marrow precursors (beyond promyelocytes) contained multiple large azurophilic granules. There were also giant granules in eosinophils, basophils, melanocytes, renal tubules, thyroid, and neurones, but not lymphocytes or monocytes. His clinical course included recurrent (ultimately fatal) infections and severe neurologic impairment. Immunofluorescent staining with fluoroscein- and rhodamine-conjugated antisera to primary and secondary granule markers showed virtually all of the granulocyte granules and rare monocyte granules to be fusion products containing both markers. Electron microscopy showed the granules to be large peroxidase-containing lysosomes. Only rare normal primary and secondary granules were present. Superoxide generation in response to opsonized zymosan was 7.3 nmole/min/10(6) cells (control 8.9); but in response to phorbol myristate acetate, only 2.2 (control 9.4). Nitroblue tetrazolium slides showed 3+ dye reduction in response to opsonized zymosan by 90% of granulocytes (control 91%) and to phorbol myristate acetate by 22% (control 99%), with 71% producing only a minimal 1+ response. Cellular contents of myeloperoxidase and beta- glucuronidase were elevated, but the percent release during exocytic degranulation was equivalent to control. Ingestion of complement- opsonized Staphylococcus aureus and zymosan was also normal. Killing of Staphylococcus aureus was 60% at 90-min incubation (control 92%). Granulocyte cyclic adenosine monophosphate (AMP) content was 4 pmole/10(7) cells (control 3.1). In order to determine whether these characteristics derived from the cells' genetic program or their environment, the patient's bone marrow was grown in long-term culture. Granulocytes produced in vitro demonstrated the same morphology, same defect in activation of nitroblue tetrazolium reduction, and same normal cyclic AMP level as those harvested from peripheral blood. These studies describe a new disorder of granulocytes; the structural similarity to, but biochemical differences from, Chediak-Higashi disease indicate the probable heterogeneity of mechanisms for the same morphological abnormality.     

Undulating tongue in Wilson's disease

We report an unusual occurrence of involuntary movement involving the tongue in a patient with confirmed Wilson''s disease (WD). She manifested with slow, hypophonic speech and dysphagia of 4 months duration, associated with pseudobulbar affect, apathy, drooling and dystonia of upper extremities of 1 month duration. Our patient had an uncommon tongue movement which was arrhythmic. There was no feature to suggest tremor, chorea or dystonia. It might be described as athetoid as there was a writhing quality, but of lesser amplitude. Thus, the phenomenology was uncommon in clinical practice and the surface of the tongue was seen to “ripple” like a liquid surface agitated by an object or breeze. Isolated lingual dyskinesias are rare in WD. It is important to evaluate them for WD, a potentially treatable disorder.     

Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex

The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.     

First identification of a diuretic,hydrochlorothiazide, in hair: Application to a doping case and interpretation of the results

An athlete contested an adverse analytical finding involving hydrochlorothiazide, and requested our laboratory for testing his hair. As there is no reference in the literature about identification of hydrochlorothiazide in hair, several volunteers were first enrolled (4 after a single 25 mg administration and 10 with daily therapeutic treatment). A specific method was developed by ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS. Hair samples were decontaminated with dichloromethane and 30 mg were incubated in buffer at pH 7.0 for 15 hours at 50°C. Then, 5 mL ethyl acetate was added for extraction. Linearity was observed for hydrochlorothiazide concentrations ranging from 5 to 2000 pg/mg. The limit of quantification was 5 pg/mg. The coefficients of variation (CVs) of repeatability and matrix effect were lower than 20%. Analysis of the 0–2‐cm segment of the 4 volunteers having received a single dose, collected 1 month after administration, was negative at the limit of quantification. The hair of the 10 patients (proximal 2 cm) on daily treatment was positive with concentrations ranging from 12 to 1845 pg/mg, with no correlation between daily dose and concentration. The athlete's hair tested positive for hydrochlorothiazide at 36 pg/mg in the segment corresponding to the period of the urinary control. Since a single exposure to hydrochlorothiazide is not detectable in hair and based on the results of the patients on daily treatment, the concentration found in the athlete has been interpreted as corresponding to repeated exposures, where it was not possible to establish the dosage and the frequency.     

LUPUS NEPHRITIS-A CRITICAL ANALYSIS OF 17 PATIENTS

Over a 3 year period from June 94 to June 97, out of 28 patients of systemic lupus, 17 were diagnosed as renal lupus. Demographic data showed 12 females and 5 males, mean age being 32.2 years (range 12 to 54 years). Mean time gap between presentation and definitive diagnosis was 32.4 days (7 days to 5 years). 2 patients (11.76%) presented renal lupus, one (5.88%) with acute interstitial lung disease and the remaining had the usual systemic manifestations of lupus. Anti dsDNA antibodies were positive in all patients while ANA was negative in 3 cases. Renal involvement consisted of rapidly progressive glomerulonephritis in 2 patients (11.76%), nephrotic syndrome in 4 (23.52%) and non nephrotic range proteinuria in 11 (64.70%) patients. Mean serum creatinine at presentation was 2.4mg/dl (0.8mg/dl to 8.9 mg/dl). Three patients were dialysis dependent. Renal histology on light microscopy comprised of class II lesions in one (5.88%), class III in 4 (23.52%), class IV in 11 (64.70%-including one with crescents) and class V in one (5.88%) patient. All patients with advanced class III/IV lesions were treated with corticosteroids and cyclophosphamide pulses. Except one patient who died of pyopericardium all others improved and their serum creatinine stabilised around 2.3 mg/dl (0.8 to 4.6 mg/dl). The study highlights the importance of early diagnosis and aggressive management in this potentially treatable disease.KEY WORDS: Lupus nephritis, Pulse cyclophosphamide