Associations between lying behavior and activity and hypocalcemia in grazing dairy cows during the transition period.

Hypocalcemia is a common metabolic disorder of transition dairy cows that is considered a gateway disease, increasing the risk of other health disorders and reducing cow performance. Clinical milk fever is associated with long periods of recumbency, and it is plausible that cows experiencing non-paretic hypocalcemia may spend more time lying; hence, lying behavior and activity measures may be useful in identifying at-risk cows. The objective of this study was to describe associations among blood calcium (Ca) status at calving and lying behavior and activity measures during the transition period in grazing dairy cows. Blood was sampled on the day of calving (d 0), and d 1, 2, 3, and 4 postcalving, and analyzed for total plasma Ca concentration. Twenty-four multiparous Holstein-Friesian and Holstein-Friesian × Jersey grazing dairy cows were classified, retrospectively, as clinically hypocalcemic (CLIN; blood Ca ≤ 1.4 mmol/L at 1 or more consecutive samplings within 48 h postcalving, but without parturient paresis). These cows were pair-matched (using milk production potential from their estimated breeding value for milk protein, mean body weight at wk -5 and -6 precalving, and, where possible, parity) with 24 cows classified as subclinically hypocalcemic (SUB; blood Ca > 1.4 and < 2.0 mmol/L at 2 consecutive samplings within 48 h postcalving), and 24 cows classified as normocalcemic (NORM; blood Ca ≥ 2.0 mmol/L at 3 consecutive samplings within 72 h postcalving). Lying behavior and activity were monitored using triaxial accelerometers from -21 to +35 d relative to calving. Data were summarized to calculate daily lying time (h/d), daily number of lying bouts (LB; no./d), mean LB duration (min/bout), and the number of steps taken (steps/d). On d 0, the CLIN group were less active and spent approximately 2.6 h longer lying than the SUB and NORM groups, particularly between 0200 and 1400 h. On d 0, the NORM group had fewer LB (16.3/d) than the SUB and CLIN groups (18.2 and 19.2/d, respectively). These differences in behavior were no longer detected 2 d postcalving, and no further differences were observed. The day before calving, the CLIN group spent 1.4 h longer lying down than did the SUB and NORM groups. Further, the relative change in steps from a precalving baseline period (d -14 to -7) until d 0 was positively, linearly associated with blood Ca concentration within 24 h postcalving. Future work should consider daily and temporal changes in behavior in individual cows to determine the potential for these measures to allow early detection of hypocalcemia.

Graduate Student Literature Review: Evaluating the appropriate use of wearable accelerometers in research to monitor lying behaviors of dairy cows.

Until recently, animal behavior has been studied through close and extensive observation of individual animals and has relied on subjective assessments. Wearable technologies that allow the automation of dairy cow behavior recording currently dominate the precision dairy technology market. Wearable accelerometers provide new opportunities in animal ethology using quantitative measures of dairy cow behavior. Recent research developments indicate that quantitative measures of behavior may provide new objective on-farm measures to assist producers in predicting, diagnosing, and managing disease or injury on farms and allowing producers to monitor cow comfort and estrus behavior. These recent research developments and a large increase in the availability of wearable accelerometers have led to growing interest of both researchers and producers in this technology. This review aimed to summarize the studies that have validated lying behavior derived from accelerometers and to describe the factors that should be considered when using leg-attached accelerometers and neck-worn collars to describe lying behavior (e.g., lying time and lying bouts) in dairy cows for research purposes. Specifically, we describe accelerometer technology, including the instrument properties and methods for recording motion; the raw data output from accelerometers; and methods developed for the transformation of raw data into meaningful and interpretable information. We highlight differences in validation study outcomes for researchers to consider when developing their own experimental methodology for the use of accelerometers to record lying behaviors in dairy cows. Finally, we discuss several factors that may influence the data recorded by accelerometers and highlight gaps in the literature. We conclude that researchers using accelerometers to record lying behaviors in dairy cattle should (1) select an accelerometer device that, based on device attachment and sampling rate, is appropriate to record the behavior of interest; (2) account for cow-, farm-, and management-related factors that could affect the lying behaviors recorded; (3) determine the appropriate editing criteria for the accurate interpretation of their data; (4) support their chosen method of recording, editing, and interpreting the data by referencing an appropriately designed and accurate validation study published in the literature; and (5) report, in detail, their methodology to ensure others can decipher how the data were captured and understand potential limitations of their methodology. We recommend that standardized protocols be developed for collecting, analyzing, and reporting lying behavior data recorded using wearable accelerometers for dairy cattle.

Associations between peripartum lying and activity behaviour and blood non-esterified fatty acids and ß-hydroxybutyrate in grazing dairy cows.

During early lactation, most dairy cows experience negative energy balance (NEB). Failure to cope with this NEB, however, can place cows at greater risk of developing metabolic disease. Our objective was to characterise, retrospectively, lying behaviour and activity of grazing dairy cows grouped according to blood non-esterified fatty acids (NEFAs) and ß-hydroxybutyrate (BHB) as indicators of postpartum metabolic state. Blood was sampled weekly for up to 4 weeks precalving, on the day of calving (day 0), daily between 1 and 4 days postcalving, and then at least weekly between week 1 and week 5 postcalving for analysis of plasma NEFAs and BHB concentrations. Two hundred and forty-four multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows were classified into one of three metabolic status groups based on maximum blood NEFAs and BHB concentrations during week 1 and 2 postcalving. A cow was classified as having either: (1) low NEFAs and low BHB (Lo-Lo; n = 78), when all blood samples were <1.0 mmol/L for NEFAs and ≤1.0 mmol/L for BHB during the first 2 weeks postcalving; (2) high NEFAs and low BHB (Hi-Lo; n = 134), when blood NEFAs were ≥1.0 mmol/L and blood BHB was ≤1.0 mmol/L at the same sampling time point during the first 2 weeks postcalving; or (3) high NEFAs and high BHB (Hi-Hi; n = 32), when blood NEFAs were ≥1.0 mmol/L and blood BHB was ≥1.2 mmol/L at the same sampling time point during the first 2 weeks postcalving. Accelerometers (IceTag or IceQube devices; IceRobotics Ltd.) were used to monitor lying and activity behaviours peripartum (-21 to +35 days relative to calving). Changes in lying behaviour and activity occurred before the mean day that cows were classified Hi-Hi and Hi-Lo (2.2 and 3.5 d postcalving, respectively). Up to 3 weeks preceding calving, Hi-Hi cows were more active, had fewer daily lying bouts (LBs), and spent less time lying than Lo-Lo cows. In addition, Hi-Hi cows had fewer daily LBs and were less active up to 4 weeks postcalving than Lo-Lo cows, but these differences were biologically small. Groups of grazing cows classified as experiencing a more severe metabolic challenge behave differently up to 3 weeks precalving than their herdmates with lower blood NEFAs and BHB postcalving. These altered behaviours may allow identification of individual cows at risk of a metabolic challenge, but further research is required.

Bacterial mutagenicity testing of urine from rats dosed with 2-ethylhexanol derived plasticizers.

Di-(2-ethylhexyl)phthalate (DEHP) produced hepatocellular carcinomas in rodents at high doses in a NTP/NCI bioassay. DEHP has not shown evidence of genotoxic activity in in vitro mutagenicity tests. We extended these studies by examining the mutagenicity of urine from rats dosed with DEHP, 2-ethylhexanol (2-EH), and several other 2-EH derived plasticizers, i.e. di-(2-ethylhexyl)adipate (DEHA), di-(2-ethylhexyl)terephthalate (DEHT) and tri-(2-ethylhexyl)trimellitate (TEHT). A modified Ames Salmonella/microsome assay was used to determine mutagenicity. Urine was pooled from male Sprague--Dawley rats dosed daily for 15 days with 2000 mg/kg of each test substance with the exception of 2-EH which was given at 1000 mg/kg. Direct plating procedures were used to determine the presence of mutagens in urine. Urine from rats dosed with 8-hydroxyquinoline was used as a positive control. There was no evidence that mutagenic substances were excreted in the urine by rats dosed with either DEHP, DEHA, DEHT, TEHT or 2-EH as determined in the presence or absence of rat liver microsomes, and with or without treatment with beta-glucuronidase/aryl sulfatase. Our findings indicate that the above test compounds were not converted to urinary metabolites that were mutagenic. These observations provide no evidence for a genotoxic mechanism for DEHP carcinogenicity in rodents.

Methods for measuring mutagenicity in urine of rats dosed with [14C]di(2-ethylhexyl)phthalate.

Di(2-ethylhexyl)phthalate (DEHP) is extensively used as a plasticizer for vinyl plastic articles. It has been found to be positive in an NCI rodent bioassay but has generally given negative results in in vitro genotoxicity tests. We therefore decided to test the urine of rats fed [14C]DEHP for mutagenic activity in the Ames Salmonella test. The recovery of radioactivity from the urine of rats dosed with [14C]DEHP was examined by solvent extraction and XAD-2 resin absorption procedures. Both of these procedures were inadequate for quantitative recovery of urinary metabolites required for subsequent mutagenicity testing using the Ames Salmonella/microsome procedure. Recoveries of less than 5% were observed using standard solvent extraction techniques whereas the XAD-2 adsorption technique gave about 67% at high resin/urine ratios. Treatment of the urine with beta-glucuronidase/aryl sulfatase did not affect these recoveries. The direct urine plating procedure represents a viable alternative to the above concentration procedures for this phthalate ester. The effects of L-histidine and the beta-glucuronidase/aryl sulfatase preparation on the background reversion frequencies of the Ames tester strains is discussed.

The relationship between growth and reversion in the Ames Salmonella plate incorporation assay.

Growth curves of the 5 commonly used Ames Salmonella tester strains have been measured turbidimetrically in semi-solid agar. Lag times, doubling times and maximum cell densities have been calculated for each of the 5 strains. The time dependence of reversion has been studied in the standard plate incorporation assay using 1-h pulsed doses of (a) bromoethane, a volatile chemical mutagen, and (b) 1-h exposures to visible light. Essentially no reversion takes place during the first 4 h after plating. Reversion is detectable between hours 4 and 16. The cumulative or integrated revertants versus time curve has the characteristics of a growth curve. Conversely the derivatives of the growth curves resemble the curves obtained in the pulsed mutagenicity studies. Thus, the reversion rate in any given 1 h interval is proportional to the growth rate during that same interval. These results suggest that mutagenic chemicals must be present during the bacterial growth cycle (about 4-16 h after plating) in order to revert the tester strains. Short-lived chemical mutagens, then, should produce enhanced results if plated 6-8 h after the bacteria. We have confirmed this for N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 9-aminoacridine and 2-aminoanthracene (with S9).

A procedure for the quantitative measurement of the mutagenicity of volatile liquids in the Ames Salmonella/microsome assay.

We have designed a closed, inert incubation system for testing the mutagenicity of volatile compounds. The containment properties of this system have been investigated using carbon-14 labelled 1,2-dibromoethane. The recovery of this solvent was about 95% following a 48-h incubation at 37 degrees. Using the Ames Salmonella/microsome mutagenicity assay we have determined the mutagenic potency of 10 common halogenated alkane solvents. Of these 10 compounds, only 1,2-dibromoethane and 1-bromo-2-chloroethane give positive results in the standard test procedure, whereas 7 of the 10 give positive results in the closed system. The specificity observed for reversion of the tester strains and the lack of any significant effect of added rat-liver "S9" fractions suggest that these haloalkanes are direct-acting "base-pair" type mutagens. The mutagenic potencies of the 7 positive compounds range from 0.001 revertants per nanomole for 1,2-dichloroethane to 0.172 revertants per nanomole for 1,2-dibromoethane. A minimum or threshold response level for each material has been calculated.

Metabolic profile of pancreatic acinar and islet tissue in culture.

BACKGROUND: The amount and condition of exocrine impurities may affect the quality of islet preparations, especially during culture. In this study, the objective was to determine the oxygen demand and viability of islet and acinar tissue post-isolation and whether they change disproportionately while in culture. METHOD: We compared the oxygen consumption rate (OCR) normalized to DNA (OCR/DNA, a measure of fractional viability in units of nmol/min/mg DNA), and the percent change in OCR and DNA recoveries between adult porcine islet and acinar tissue from the same preparation (paired) over 6-9 days of standard culture. Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture. RESULTS: The mean (±SE) OCR/DNA was 74.0 ± 11.7 units higher for acinar (vs islet) tissue on the day of isolation (n = 16, P < .0001), but 25.7 ± 9.4 units lower after 1 day (n = 8, P = .03), 56.6 ± 11.5 units lower after 2 days (n = 12, P = .0004), and 65.9 ± 28.7 units lower after 8 days (n = 4, P = .2) in culture. DNA and OCR recoveries decreased at different rates for acinar versus islet tissue over 6-9 days in culture (n = 6). DNA recovery decreased to 24 ± 7% for acinar and 75 ± 8% for islets (P = .002). Similarly, OCR recovery decreased to 16 ± 3% for acinar and remained virtually constant for islets (P = .005). CONCLUSION: Differences in the metabolic profile of acinar and islet tissue should be considered when culturing impure islet preparations. OCR-based measurements may help optimize pre-islet transplantation culture protocols.

Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation.

BACKGROUND: Replacement of ß-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36-72-hour culture period before IT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability. METHODS: After standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C, whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IEQ)/cm(2) adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content [OCR/DNA] and membrane integrity). RESULTS: Results indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin in the culture media. CONCLUSIONS: Current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability before transplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations.

Human islet viability and function is maintained during high-density shipment in silicone rubber membrane vessels.

BACKGROUND: The shipment of human islets (IE) from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment). METHOD: IE were isolated from two manufacturing centers and shipped in 10-cm(2) surface area SRM vessels in temperature- and pressure-controlled containers to a distant center after at least 2 days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a microcentrifuge tube negative control (NC). LD was designed to mimic the standard culture density for IE preparations (200 IE/cm(2)), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1-3 vessels. Upon receipt, islets were assessed for viability (measured by oxygen consumption rate normalized to DNA content [OCR/DNA)]), quantity (measured by DNA), and, when possible, potency and function (measured by dynamic glucose-stimulated insulin secretion measurements and transplants in immunodeficient B6 Rag(+/-) mice). Postshipment OCR/DNA was not reduced in HD vs LD and was substantially reduced in the NC condition. HD islets exhibited normal function postshipment. Based on the data, we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function.

Hypothermic Perfusion Preservation of Pancreas for Islet Grafts: Validation Using a Split Lobe Porcine Model.

The demand for high-quality islets for transplantation in type I diabetics will increase as the current clinical trials transition into standard of care. The mode of preservation of donor pancreata is critical to this mission since islets are very sensitive to ischemic injury. Hypothermic perfusion preservation (HPP) is being investigated for extended pancreas preservation in light of the beneficial effects reported for other organs. The present pilot study aimed to establish the potency of porcine islets isolated from pancreata after 24 h of HPP at 4-8°C. The study design included a split-lobe pancreas model that permitted paired comparisons of islets isolated from 24-h HPP splenic lobes with nonperfused, fresh control duodenal/connecting lobes stored at 4°C for <3 h. Prior to transplantation, islet viability was assessed in vitro using the ratio of oxygen consumption rate to DNA (OCR/DNA) assay and correlated with subsequent in vivo function by transplantation in diabetic immunodeficient mice. The OCR/DNA (mean ± SD) measured after 7 days of culture and immediately prior to transplantation for islets from the 24-h HPP group was 269 ± 19 nmol/min/mg DNA, which was higher but not statistically different to the mean of 236 ± 43 for the counterpart control group. All four nude mice transplanted with islets from the 24-h HPP group showed diabetes reversal, compared with five of six transplants from the control group. In conclusion, islets isolated from adult porcine pancreata after 24-h HPP exhibited high viability as measured by OCR/DNA and were able to consistently reverse diabetes in a nude mouse bioassay.

T cell cytotoxicity in cystic fibrosis: relationship to pulmonary status.

T cell cytotoxicity (CTL) to an allogeneic lymphocyte target was evaluated in patients with cystic fibrosis (CF) before and during pulmonary exacerbations (group 1) compared to another group of CF patients who had stable pulmonary disease activity during their two study periods (group 2). CTL activity was significantly decreased in group 1 subjects studied prior to their pulmonary flares and in group 2 CF patients compared to normal controls at effector:target ratios of 12.5:1 and 6.25:1 (p less than 0.05 and p less than 0.05, respectively). Furthermore, in group 1, CTL lysis was significantly decreased during pulmonary flares compared to before flares at the 25:1 and 12.5:1, effector:target, (p less than 0.05 and p less than 0.05, respectively). T suppressor cell activity as measured by effect on in vitro control B cell IgM synthesis was significantly increased during pulmonary flares (p less than 0.05). Diminished CTL may be partially responsible for persistent colonization of Pseudomonas aeruginosa in CF.

Immune response in experimentally induced uremia. VI. Uremic macrophages are defective in their ability to present antigen to T cells.

The ability of uremic lymphocytes to respond to antigens was examined. We have found that (1) The ability of uremic unfractionated lymph node cells to respond to antigens is severely diminished when compared to the response of control cells; (2) Uremic macrophages are defective in their ability to present antigen to T cells; (3) Treatment of control splenic macrophages with monoclonal anti-Ia antibodies diminishes these macrophages' ability to present antigen, while uremic macrophages so treated show little change in their already diminished accessory cell function; and (4) The uremic splenic macrophage population has more small cells and less Ia determinants per cell than do control splenic macrophages as determined by cytofluorographic analysis. The percentage of Ia+ splenic macrophages is similar in control and uremic rats. It appears that the diminished response of uremic cells to antigens is due to the inability of uremic macrophages to present antigen to T cells. This may play a role in the increased rate of infections seen in uremic patients.

Suppression of mixed lymphocyte culture by uremic adherent spleen cells and peritoneal macrophages is dependent on a cyclophosphamide-sensitive cell.

A model of experimentally induced uremia in the rat has been used to study the effect of uremia on the response of spleen cells to alloantigens. The proliferative ability of uremic spleen cells in mixed lymphocyte culture is significantly suppressed when compared to that of cells from control animals. This suppression appears to be due to both adherent suppressor cells which can be eliminated by adherence to rayon wool and to the inability of uremic T cells to respond to alloantigens. In addition, unstimulated peritoneal macrophages ( PMO ) obtained from uremic rats were also shown to be suppressive to the response of control spleen cells to alloantigens. The suppression by uremic adherent spleen cells and PMO is regulated by cyclophosphamide-sensitive cells.

Immune response in experimentally induced uremia. IV. Characterization of suppressor peritoneal macrophages in the uremic rat.

The effect of unstimulated uremia PM phi on the response of syngeneic control NA spleen cells to mitogens was examined. Nonstimulated PM phi purified from uremic rats were found to be significantly more suppressive than similar numbers of control PM phi. It was found that indomethacin treatment, as well as anti-la treatment, only partially reverses the suppressive activity of uremic PM phi as compared to control PM phi, thus indicating that uremic suppressor PM phi have different characteristics from control PM phi. In addition, uremic PM phi suppress via a suppressor factor released to the supernatant over 24 hr incubation. The enhanced suppressor activity of uremic PN phi as well as their different characteristics may have relevance to the severely suppressed cell-mediated immunity observed in uremia.

Serum anti-Aspergillus fumigatus antibodies by immunoblot and ELISA in cystic fibrosis with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) occurs with a prevalence of 5% to 15% in patients with cystic fibrosis (CF). Because of the frequent colonization with Aspergillus fumigatus (Af) in CF, the causative agent of ABPA, antibody reactivity to Af proteins is frequently observed, which obscures the diagnosis of ABPA. Patients with CF are also categorized according to the presence of positive skin test responses to Af and/or the presence of positive precipitins. In this study we used ELISA and immunoblot assay to detect IgE and IgG anti-Af antibodies in patients with CF and ABPA (n = 13) compared with other groups of patients with CF: those with positive skin test and positive precipitin results (n = 18), those with positive skin test and negative precipitin results (n = 14), those with negative skin test and positive precipitin results (n = 10), and those with negative skin test and negative precipitin results (n = 35). IgE and IgG anti-Af antibodies were significantly elevated in patients with ABPA as determined by both immunoblot assay (p < 0.01) and ELISA (p < 0.01). However, detection of Af antibodies by ELISA was more sensitive in discriminating patients with CF and ABPA from patients with CF who had positive skin test and positive precipitin results but lacked radiographic and clinical evidence of ABPA. In patients with CF and ABPA the immunoblot assays demonstrated a multitude of IgE, IgG, and IgA antibody responses to Af proteins, which ranged in molecular weight from 14 kd to greater than 106 kd. The level of IgE anti-Af antibody to individual proteins decreased during remissions of ABPA.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of ethylene oxide allergy in sterile shunt malfunctions.

Failure of an intact ventriculoperitoneal shunt, in the absence of an overt infection, is often due to its occlusion by cellular debris and/or an abdominal pseudocyst. This failure is thought to be caused by an infection by an organism which is difficult to culture or by some poorly defined allergic response to the shunt materials. Little attention has been directed to the treatment that the shunts receive prior to implantation: specifically, their exposure to ethylene oxide as a means of sterilization. We have found ethylene oxide metabolites in the spinal fluid of children with shunt malfunction months after their systems were implanted. Many of these patients had coincident CSF eosinophilia. In addition, two of the children had detectable serum IgE antibody directed against an albumin-ethylene oxide conjugated protein. Both of these children had several shunt malfunctions within a short period, yet neither child could be shown to have a shunt infection despite multiple cultures. We therefore suggest that in some patients proteins altered by ethylene oxide incite an IgE mediated response which may lead to shunt malfunction.

Serum immunoglobulins E and G anti-Aspergillus fumigatus antibody in patients with cystic fibrosis who have allergic bronchopulmonary aspergillosis.

Patients with cystic fibrosis frequently have pulmonary colonization with Aspergillus fumigatus (Af) and develop anti-Af immunoglobulin E (IgE) and IgG antibodies. The diagnosis of allergic bronchopulmonary aspergillosis in subjects with cystic fibrosis is difficult because of the high incidence of Af colonization, with development of humoral antibody responses. In this study, we sequentially measured serum anti-Af IgE (Af-E) and IgG (Af-G) antibodies by ELISA in subjects with cystic fibrosis. In subjects with cystic fibrosis who have allergic bronchopulmonary aspergillosis, Af-E and Af-G antibodies were significantly increased when compared with other groups of patients with cystic fibrosis who had positive skin tests or precipitins to Af (or both) (p less than 0.01, p less than 0.01, respectively). In addition, increased Af-E and Af-G levels were sometimes seen in other groups, especially subjects with cystic fibrosis who had positive Af skin tests or precipitin tests, two of whom later developed criteria diagnostic of allergic bronchopulmonary aspergillosis. Thus, serum Af-E and Af-G levels were quantitatively increased in subjects with cystic fibrosis who had allergic bronchopulmonary aspergillosis and thus adjunctive data in diagnosis. However, it also suggested that subclinical pulmonary inflammation may also occur.

T- and B-cell dysregulation of IgE synthesis in cystic fibrosis patients with allergic bronchopulmonary aspergillosis.

Since Aspergillus fumigatus (Af)-specific and polyclonal serum IgE levels are characteristically elevated in patients with allergic bronchopulmonary aspergillosis (ABPA), we evaluated in vitro regulation of IgE synthesis in cystic fibrosis (CF) patients with ABPA. We studied 11 CF patients with ABPA, 37 patients with positive Af prick skin tests and/or IgG precipitating antibodies (ST/PPT+), and 35 patients with no humoral or skin responses to Aspergillus (ST/PPT-). Mean serum IgE concentration was significantly elevated in CF subjects with ABPA compared to ST/PPT+ and ST/PPT- patients, 2866 vs 303 and 61 IU/ml, respectively (P less than 0.01). In vitro studies demonstrated that ABPA patients' B cells spontaneously synthesized significantly increased amounts of IgE compared to ST/PPT positive and negative subjects, 1980 vs 220 and 13 pg/ml, respectively (P less than 0.01). In addition, preformed B-cell-associated IgE was also significantly elevated in ABPA subjects (P less than 0.01), indicating prior in vivo activation. Supernatant cultures of Af-stimulated T cells from ABPA subjects significantly induced allogeneic B-cell IgE synthesis compared to ST/PPT positive and negative CF subjects, 206 vs 13 and 4 pg/ml, respectively (P less than 0.01). Thus T cells stimulated with Aspergillus antigens secrete cytokines that induce B-cell IgE synthesis in ABPA subjects. B-cell IgE hyperactivity is manifested by in vivo and in vitro increased IgE concentrations. Analyses of T-cell regulation and B-cell IgE synthesis distinguish CF subjects with ABPA from Aspergillus sensitive non-ABPA subjects.

Abnormal in vitro thymocyte differentiation in a patient with severe combined immunodeficiency-Nezelof's syndrome.

An in vitro coculture model system of CD34+ stem cells and allogenic cultured thymic epithelia fragments was used to evaluate thymocyte differentiation in a 9-month-old child of Amish descent with Nezelof syndrome. Though the patient's stem cells differentiate to acquire normal expression of CD2 and CD7, later steps of maturation were abnormal. There was detectable but reduced expression of CD3 and CD4 phenotypes. CD44+ expression, however, was markedly reduced. CD44 is an adhesion molecule, interacting with the matrix ligands hyaluronan and fibronectin, and is expressed early in thymocyte differentiation and subsequently in mature T cells. It is hypothesized that abnormal expression of CD44 in a variant of severe combined immunodeficiency, Nezelof's syndrome, interferes with normal thymocyte and thymic epithelial interaction, which leads to abnormal thymocyte differentiation.