Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA).

Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and HP2), therefore three genotypes are present in the population, i.e., HP1-1, HP2-1, and HP2-2. A biochemical role for nascent haptoglobin 2 (pre-haptoglobin 2 or pre-HP2) as the only known modulator of intestinal permeability has been established. In addition, elevated levels of serum pre-HP2 have been detected in multiple conditions including celiac disease and type I diabetes, which are believed to result in part through dysregulation of the intestinal barrier. In this study, we report the development of a monoclonal antibody that is specific for pre-HP2 with a binding affinity in the nanomolar range. Additional antibodies with specificities for preHP but not mature haptoglobin were also characterized. A sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated. The ELISA showed high specificity for pre-HP2 even in the presence of excess pre-HP1 or mature haptoglobins, and has excellent linearity and inter- and intra-assay reproducibility with a working range from 3.1ng/mL to 200ng/mL. Testing of sera from 76 healthy patients revealed a non-Gaussian distribution of pre-HP2 levels with a mean concentration of 221.2ng/mL (95% CI: 106.5-335.9ng/mL) and a median value of 23.9ng/mL. Compared to current approaches, this ELISA offers a validated, monoclonal-based method with high sensitivity and specificity for measuring pre-HP2 in human serum.

The pharmacological chaperone 1-deoxygalactonojirimycin increases alpha-galactosidase A levels in Fabry patient cell lines.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene encoding alpha-galactosidase A (alpha-Gal A), with consequent accumulation of its major glycosphingolipid substrate, globotriaosylceramide (GL-3). Over 500 Fabry mutations have been reported; approximately 60% are missense. The iminosugar 1-deoxygalactonojirimycin (DGJ, migalastat hydrochloride, AT1001) is a pharmacological chaperone that selectively binds alpha-Gal A, increasing physical stability, lysosomal trafficking, and cellular activity. To identify DGJ-responsive mutant forms of alpha-Gal A, the effect of DGJ incubation on alpha-Gal A levels was assessed in cultured lymphoblasts from males with Fabry disease representing 75 different missense mutations, one insertion, and one splice-site mutation. Baseline alpha-Gal A levels ranged from 0 to 52% of normal. Increases in alpha-Gal A levels (1.5- to 28-fold) after continuous DGJ incubation for 5 days were seen for 49 different missense mutant forms with varying EC(50) values (820 nmol/L to >1 mmol/L). Amino acid substitutions in responsive forms were located throughout both structural domains of the enzyme. Half of the missense mutant forms associated with classic (early-onset) Fabry disease and a majority (90%) associated with later-onset Fabry disease were responsive. In cultured fibroblasts from males with Fabry disease, the responses to DGJ were comparable to those of lymphoblasts with the same mutation. Importantly, elevated GL-3 levels in responsive Fabry fibroblasts were reduced after DGJ incubation, indicating that increased mutant alpha-Gal A levels can reduce accumulated substrate. These data indicate that DGJ merits further evaluation as a treatment for patients with Fabry disease with various missense mutations.

Limited ARSAC licence acquisition for radionuclide radiology.

Radionuclide radiology faces a potentially crippling future manpower shortage. A combination of future retirement, few trainees and many currently unfilled posts threatens to limit future service delivery. The case is made for in-house modular training of existing consultants as the way forward for radionuclide radiology, allowing limited ARSAC licence acquisition. We propose this as a viable solution to the manpower problem in radionuclide radiology.

Differential bore sheath breast biopsy--a pilot study of a new technique.

The increasing requirement for core biopsy in the diagnosis of early breast disease puts additional demands on radiologists' time and expertise. We present a new approach to percutaneous breast biopsy of impalpable lesions with core biopsy and simultaneous hook wire localization. The technique allows accurate localization of breast lesions and is unique in that it facilitates simultaneous hook wire localization. Percutaneous biopsy by this method was accurate in 9 of 11 patients.

Local staging of prostate cancer with 0.2 T body coil MRI.

This study was undertaken to evaluate the accuracy of low field strength body coil MRI in the staging of clinically localized prostate cancer. Fifty-three patients with prostate cancer were examined on a 0.2 T body coil system before undergoing radical prostatectomy. Of the 20 cases with unconfined stage T3 disease on histology, 12 were correctly staged, whilst three cases were overstaged by MRI. (Accuracy 79.2%, sensitivity 60%, and specificity 90.9%.) The accuracy, sensitivity and specificity for the detection of capsular penetration were 77.3%, 55% and 90.9%, respectively, whilst those for seminal vesical invasion were 94.3%, 83.3% and 95.7%, respectively. It is concluded that a high level of staging accuracy, comparable to that obtained in some published studies using high field strength endorectal coil MRI, can be obtained using 0.2 T body coil MRI.

Short communication: Prospective assessment of a new graduated hookwire for the localization of impalpable breast lesions.

The benefits of the addition of site specific variable graduations to a conventional mammographic localizing wire were assessed in a prospective multidistrict study. Six surgeons and five radiologists localized lesions in 31 patients in three District General Hospitals. The advantages of the graduated wire were shown to be greater ease and accuracy of radiological localization with easier surgical excision and a smaller resected breast specimen.

Testicular infarction mimicking tumour on scrotal ultrasound--a potential pitfall.

We present two cases of spontaneous testicular infarction. Although the clinical presentation indicated an acute pathology, the ultrasound examination demonstrated focal, hyporeflective intra-testicular lesions that could not be discriminated from tumour. As a result of the ultrasound examinations, radical orchidectomies were performed and diagnoses of spontaneous testicular infarction proven histologically.

Beneficial effect of cod liver oil in murine endotoxemia.

Cod liver oil (CLO), a marine fish oil, contains approximately 20% omega-3 fatty acids (OFA). When CLO is administered to humans, inhibition of platelet aggregation, decreased platelet arachidonic acid levels, and reduced levels of endotoxin-induced thromboplastin synthesis by monocytes are observed. Since endotoxin causes both increased platelet aggregation and monocyte generation of thromboplastin with resultant microvascular compromise, the purpose of this study was to determine whether CLO is protective in murine endotoxemia. Swiss Webster mice were given CLO (1.0mg, 10.0mg, or 100mg), or 100mg triolein (an unsaturated fat containing no OFA) or saline (control) intraperitoneally (IP) three hours prior to IP administration of 0.3mg E.coli endotoxin. Survivals at 48 hours post-endotoxin administration were as follows: (A) 1.0mg CLO (4/20, 20%); (B) 10mg CLO (5/20, 25%); (C) 100.0mg CLO (11/20, 55%); (D) 100mg triolein (1/20, 5%); (E) 0.13cc saline (1/20, 5%). The significance of groups A,B,C,D verses the control group E is as follows: A vs E, p = 0.15; B vs E, p = 0.08; C vs E, p = 0.0006; D vs E, p = 0.76. This study demonstrates the beneficial effect of 100mg parenteral CLO in murine endotoxemia. This effect is probably mediated through antiplatelet and/or antimonocyte activating mechanisms.

A comparison of intravenous access sites for bolus injections during shock and resuscitation after emergency room thoracotomy with and without aortic crossclamping.

Thoracotomy with aortic crossclamping is used to resuscitate trauma victims. Pharmacologic boluses are often given via intravenous lines of central, brachial, or femoral origin. This study was undertaken to determine the efficacy of intravenous access site on delivery of a bolus injection to the heart with thoracotomy and aortic crossclamping during shock and resuscitation. Six dogs were anesthetized, intubated, and underwent brachial and femoral venotomy and Swan-Ganz insertion (central). Baseline measurements of central, brachial, and femoral transit times for 10 cc cold saline were obtained via Swan-Ganz thermistor. Each animal then underwent thoracotomy, aortic crossclamping alone, hemorrhage to blood pressure (BP) 50 mm Hg for 30 minutes with aorta unclamped and then aorta crossclamped and resuscitation with lactated Ringer's Solution with aorta unclamped and then crossclamped. Femoral-Brachial Index (FBI) was determined by dividing femoral transit time by brachial transit time at each observation. The data suggest that femoral access significantly prolongs bolus transit time when compared with central or brachial access during aortic crossclamping in the euvolemic, shock, or aggressively resuscitated model. Brachial access is therefore the preferred route for bolus injection delivery in the emergency room thoracotomy with or without aortic crossclamping because it provides expedient bolus delivery equal to central access and superior to femoral access.

A "treated" model for severe hemorrhagic shock: a comparison of conventional and germ-free animals.

In an unanesthetized "treated" model of severe hemorrhagic shock, a bacteremia originating from the animal's enteric flora was demonstrated by finding radiolabeled Escherichia coli in the blood as early as 2 hr after the onset of shock. In 50 patients admitted to our trauma unit, the observation was similar, with 56% positive blood cultures, when their mean arterial blood pressure was 80 mmHg or less. The relationship of bacterial translocation and the high post-shock mortality in our conventional (CV) animal model is being evaluated in germfree (GF) Sprague-Dawley rats. Preliminary observations in 15 GF animals showed 80% survival at 24 hr post-shock, 54% at 48 hr, and 42% at 72 hr compared with those of CV animals in previous experiments with survival at similar times of 73%, 20% and 7%, respectively.

Does the bacteremia observed in hemorrhagic shock have clinical significance? A study in germ-free animals.

We have recently reported the rapid appearance of bacteria and endotoxin in the blood of rats and of trauma patients in the course of 30 minutes to 2 hours of hemorrhagic shock. The current study was designed to determine the effect of this bacteremia and endotoxemia on survival. Thirty-three conventional (C:group 1) and 36 germ-free (GF:group 2) Sprague Dawley rats were subjected to our previously described model of treated hemorrhagic shock. Survival in the GF group was significantly better than the C group at 24, 48, and 72 hours after shock. Endotoxin levels were elevated in 88% of C group during shock and in 28% of GF group. The gut of the GF animal contains endotoxin (26 ng/gm of stool) as does the sterile food supply (393 ng/gm of rat chow).

Endotoxemia and bacteremia during hemorrhagic shock. The link between trauma and sepsis?

Previous investigations of a treated model of hemorrhagic shock in the rat indicated the frequent occurrence of bacteremia that appeared to derive from the gut. This paper determines the incidence of bacteremia and endotoxemia during the acute shock period and compares this with similar observations in humans in varying degrees of shock. Studies in 26 rats indicated that bacteremia and endotoxemia was present in 50% and 87%, respectively, by the end of 2 hours at a mean arterial pressure of 30 mmHg. Observations in 50 patients admitted to the trauma unit showed that positive bacterial blood cultures were present in 56% when the admission systolic blood pressure was 80 mmHg or less (p less than 0.01 compared with either of the other groups). Endotoxemia was noticed in two such patients. Direct access of bacteria and endotoxin to the blood stream may occur during hemorrhagic or traumatic shock and is the probable cause of subsequent sepsis in traumatized patients when no other source is apparent.

Effect of a butter-rich diet on murine frostbite.

A simple reproducible, humane model for producing frostbite in rodents is desirable. We have developed such a model and have investigated the effects of a butter-rich diet on severe cold injury. Mice (25 +/- 2 g) were randomly divided into two groups (n = 10). One group was given a standard rodent diet and the other group a butter-enriched (25%) rodent diet for 1 month. Under pentobarbital sodium anesthesia the mice were placed in a plastic funnel with the stem removed so that the entire tail protruded. The tails were submerged in a freezing solution (-18 degrees C) for 8 min. The mice received no further treatment. Tail lengths were measured 2 weeks after freezing to determine the amount of tail surviving from cold injury after autoamputation. Mean tail lengths (cm +/- SD) for the two groups were as follows: butter-rich diet, 4.3 +/- 1.8; standard diet, 7.3 +/- 2.0 (P less than 0.002). A high saturated-fat diet has a deleterious effect in murine frostbite. This simple model can be employed to test various modalities in the treatment of frostbite.

Experimental preadmission program to encourgage home care for severely and profoundly retarded children.

An experimental treatment program was designed to reduce institutionalization of severely and profoundly retarded children under 6 years of age. Subjects were randomly assigned to experimental and control groups. Results showed that counseling with the mother alone tends to result in increased marital conflict as well as a decision to keep the child at home, particularly for parents who are uncertain about their plans and hopes for the child. Since this conflict could defeat the real purpose of fostering the welfare of the child, family counseling should be used and consensus within the family should be sought. Instructing the mother in child-training procedures tends to reduce the number of instances of objectionable child behavior, as well as to induce a decision to keep the child at home, particularly for parents who are uncertain about their hopes and plans for the child.