Human Serum Albumin Conjugates Research Articles

Near real-time biosensor-based detection of 2,4-dinitrophenol

A fluorescent biosensor assay has been developed for near real-time detection of 2,4-dinitrophenol (DNP). The assay was based on fluorescent detection principles that allow for the analysis of antibody/antigen interactions in solution using the KinExA™ immunoassay instrument. Our KinExA™ consisted of a capillary flow observation cell containing a microporous screen that maintains a compact capture antigen-coated bead bed. The bead bed was comprised of polymethylmethacrylate (PMMA) beads coated with dinitrophenol–human serum albumin (DNP–HSA) conjugate. Phosphate buffered saline (PBS) solutions, containing various concentrations of free DNP, were incubated for 30 min with mouse anti-DNP monoclonal antibody to equilibrium. Solutions containing the DNP–monoclonal antibody complex and possible excess free antibodies were then passed over DNP–HSA labeled beads. The free monoclonal anti-DNP antibody, if available, was then bound to the DNP–HSA fixed on the beads. The system was then flushed with excess PBS to remove unbound reactants in the bead bed. The beads were then subjected to brief contact with PBS solutions containing goat anti-mouse fluorescein isothiocyanate (FITC)-labeled secondary antibody, once again, followed by a short PBS flush. The fluorescence was recorded during the addition of the FITC labeled secondary antibody to the bead bed through the final PBS flushing with the KinExA™. The amount of DNP detected could then be determined from the fluorescent slopes that were generated or by the remaining fluorescence that was retained on the beads after final PBS flushing of the system. This assay has been able to detect a minimum of 5 ng/ml of DNP in solution and can be adapted for other analytes of interest simply by changing the capture antigen and antibody pairs.

Preparation and characterisation of rose Bengal-loaded surface-modified albumin nanoparticles

Surface-modified albumin nanoparticles were prepared from two poly(ethylene glycol)–human serum albumin conjugates: poly(thioetheramido acid)–poly(ethylene glycol) copolymer-grafted HSA (HSA–PTAAC–PEG) and methoxy poly(ethylene glycol)-grafted HSA (HSA–mPEG). Rose bengal (RB) was used as a model drug for encapsulation into the nanoparticles either during the particle production or by adsorption post particle preparation. The drug incorporation and release was affected by the different production methods and the different polymer compositions. When RB was loaded in HSA and HSA/HSA–PTAAC–PEG nanoparticles, up to 5% (w/w) drug content was achieved. The drug loading in HSA–mPEG nanoparticles was much lower and the results from the microcalorimetry study indicated that the low loading efficiency was due to less drug–protein binding sites available in the HSA–mPEG molecule as compared to the HSA molecule. The release of RB from the albumin nanoparticles was very slow in PBS and dramatically accelerated in the presence of trypsin. Compared with unmodified nanoparticles, the slower release of RB from the surface-modified HSA nanoparticles in the presence of the enzyme suggested that the existence of a steric hydrophilic barrier on the surface of the nanoparticles made digestion of the nanoparticles more difficult.

Diverse profiles of specific IgE response to toluene diisocyanate (TDI)-human serum albumin conjugate in TDI-induced asthma patients.

The prevalence studies on specific IgE to toluene diisocyanate (TDI)-human serum albumin (HSA) conjugate in TDI-induced asthma have shown variable results. In this study, we attempted to compare specific IgE bindings to TDI-HSA conjugate and its specificity using 3 different conjugates. Sera were collected from 20 TDI-induced asthma and 10 controls. Specific IgE were measured by ELISA using three TDI-HSA conjugates; two from Carnegie Mellon (CM; 98 and 99 CM conjugates) and one from Ajou University. To evaluate specificity and cross-reactivity, ELISA inhibition tests were applied. Positive and negative predictive values between Ajou conjugate and 98 CM conjugate were 75% and 100%. Those between Ajou and 99 CM were 100% and 93.8%. One patient showed an isolated positive response to the Ajou with negative responses to the other two conjugates. ELISA inhibition test using this patient's serum revealed the significant inhibitions by the Ajou and minimal inhibitions by the others. On the other hand, another patient showed an isolated positive response to 99 CM with negative responses to the others, and ELISA inhibition test showed significant inhibition by 99 CM with minimal inhibitions by the others. These results suggest that specific IgE bindings to a new antigenic determinant of TDI-HSA conjugate can be heterogeneous and differ from one individual to another.

Synthesis of the Lewis b hexasaccharide and squarate acid–HSA conjugates thereof with various saccharide loadings

The Lewis b hexasaccharide, α- l-Fuc p-(1→2)-β- d-Gal p-(1→3)-[α- l-Fuc p-(1→4)]-β- d-Glc pNAc-(1→3)-β- d-Gal p-(1→4)-β- d-Glc p, has been synthesised using a convergent synthesis. Starting from ethyl 4,6- O-benzylidene-2-deoxy-2-phthalimido-1-thio-β- d-glucopyranoside, a thioglycoside tetrasaccharide donor block, was constructed through two orthogonal glycosylations with glycosyl bromide donors. First, the galactose moiety was introduced using silver triflate as a promoter and then the two fucose residues under halide-assisted conditions. Finally, this tetrasaccharide was linked to a spacer-equipped 3 I,4 I-diol lactose acceptor in a DMTST-promoted coupling to give, after deprotection, the Lewis b hexasaccharide as its 2-aminoethyl spacer-equipped derivative. This was coupled to human serum albumin (HSA), using the squarate ester methodology, in various saccharide–protein ratios, to give neoglycoconjugates with different saccharide loadings in about 50% efficiency.

In vitro reactivity of penicilloyl and penicillanyl albumin and polylysine conjugates with IgE-antibody

Penicilloylated (BPO) and penicillanylated (BPA) poly- l-lysine (PLL) and human serum albumin (HSA) were prepared and characterized by penamaldate assay and proton NMR spectroscopy. The conjugates were coupled to nitrocellulose (NC) discs and cyanogen bromide activated paper discs and their in vitro reactivities with serum IgE antibodies were examined. Results showed that on paper discs, 55.3 and 83% of the sera reacted with PLL conjugates of BPO and BPA, respectively, while 41.5 and 58.1% reacted with HSA conjugates. On NC discs, HSA conjugates gave better results, 75.6 and 70.7%, respectively for BPO and BPA, compared with 38.6 and 50%, respectively for the PLL conjugates. Overall, the BPA–PLL conjugate on paper discs proved to be the most reactive preparation. Addition of the BPO–PLL paper disc preparation detected more positive sera (85.1%) and we believe that the combined use of these two specificities offers the best test for the detection of penicillin-reactive IgE antibodies.

In vitro demonstration of IgE antibody to folate-albumin in anaphylaxis from folic acid

Background: Folic acid (the synthetic form of folate B vitamins in foods) is widely used in vitamin supplements. Anaphylaxis from ingestion or injection of folic acid suggests IgE antibody–mediated mechanisms, but this has not been demonstrated previously in vitro. Objective: This study was conducted to better define the mechanism of folic acid hypersensitivity and cross-reactivity among folic acid congeners. Methods: Skin testing was performed with folic acid congeners in a woman who developed anaphylaxis after ingestion of 2 different multivitamin preparations containing folic acid. In vitro immunologic serum studies were conducted using a folate–human serum albumin (HSA) conjugate prepared by a novel application of carbodiimide condensation. Results: The patient had positive immediate-type skin test reactions to folic acid and several folate analogues including leucovorin (folinic acid). Urticaria developed during graded oral test dosing with leucovorin. Using a dot immunoblot assay or an ELISA for IgE antibody to folate-HSA, results of the patient's serum testing were positive, whereas results of sera from normal control subjects were negative, the first in vitro demonstration of IgE to a folic acid–protein conjugate. By ELISA, the positive result of the patient's serum was inhibited significantly by serum coincubation with folate-HSA, but not HSA or folic acid. Conclusions: Immediate hypersensitivity to folic acid and possibly other vitamins can be mediated by IgE antibody to conjugates formed between vitamins and self-proteins or polypeptides. Leucovorin can have clinically important immunologic cross-reactivity with folic acid. A diet rich in natural folates (pteroylpolyglutamates) appears useful as a management strategy for providing adequate nutrition to patients with folic acid hypersensitivity. {J Allergy Clin Immunol 2000;106:386-9.)

Dexamethasone coupled to albumin is selectively taken up by rat nonparenchymal liver cells and attenuates LPS-induced activation of hepatic cells

Background/Aims: The human serum albumin (HSA) conjugate Dexa 10-HSA was designed to specifically deliver the anti-inflammatory drug dexamethasone (Dexa) to nonparenchymal cells (NPC) in the rat liver. NPC play an important role in the pathogenesis of acute and chronic inflammatory liver diseases like fibrosis. Targeting Dexa to these cells might reduce its adverse effects and increase the efficacy. Methods: Tissue and intrahepatic distributions of Dexa 10-HSA were assessed in normal and fibrotic rats with 125I-labelled conjugate and by immunohistochemistry. The effect of the conjugate on lipopolysaccharide (LPS)-induced inflammation and cell activation was studied in vitro with precision-cut liver slices and in vivo. Results: Ten minutes after i.v. injection 72±13% and 65±12% of a tracer dose of Dexa 10-HSA had been taken up in normal and fibrotic livers, respectively. Unconjugated Dexa also accumulated in livers, but cellular distribution studies revealed an accumulation in parenchymal cells (NPC vs. parenchymal cell (PC) ratio=0.29±11, p<0.005) whereas Dexa 10-HSA accumulated in nonparenchymal cells (NPC/PC ratio=7.9±3.1). Both coupled and uncoupled Dexa showed effective inhibition of LPS-induced NO x and TNFα production in precision-cut liver slices. At low concentrations (0.02 μM), however, Dexa 10-HSA was more efficient at inhibiting TNFα production than uncoupled Dexa. In fibrotic rats Dexa 10-HSA (3 mg/kg) and an equimolar amount of uncoupled Dexa (0.22 mg/kg) both significantly promoted survival after LPS-induced acute inflammation. Conclusion: Dexa 10-HSA was at least as effective as uncoupled Dexa at inhibiting LPS-induced effects, which indicates that HSA-bound Dexa is pharmacologically active. Coupling Dexa to HSA shifted the accumulation of Dexa from the PC to the NPC of livers. Since mediator release from NPC is crucial in the initiation and propagation of the fibrotic process, selective delivery of Dexa in NPC may improve the efficacy and safety of corticosteroid treatment of liver fibrosis.

152 Role of specific IgE and IgG to toluene diisocyanate (TDI)-human serum albumin (HSA) conjugate as prognostic factor for TDI-asthma

152 Role of specific IgE and IgG to toluene diisocyanate (TDI)-human serum albumin (HSA) conjugate as prognostic factor for TDI-asthma

Specific IgG, but not specific IgE, antibodies to toluene diisocyanate–human serum albumin conjugate are associated with toluene diisocyanate bronchoprovocation test results

Background: The role of specific IgG to toluene diisocyanate (TDI) in the pathogenesis of TDI-induced asthma still remains unclear. Objective: We sought to evaluate the clinical significance of serum-specific IgG to TDI-human serum albumin (HSA) conjugate in subjects with TDI-induced asthma compared with specific IgE antibody. Methods: One hundred three subjects were enrolled and divided into 4 groups according to specific bronchoprovocation test (BPT) results: 50 subjects with TDI-induced asthma with positive results on TDI BPT were defined as group 1, 13 symptomatic workers exposed to TDI with negative results on TDI BPT were defined as group 2, 20 unexposed patients with allergic asthma were defined as group 3, and 20 unexposed healthy control subjects were defined as group 4. Serum-specific IgG and IgE antibodies to TDI-HSA conjugate were detected by means of ELISA. Results: The prevalence of specific IgG antibody to TDI-HSA conjugate was significantly higher in group 1 than in group 2 (46% vs 7.7%, P = .01) or group 3 (0%, P < .01). No significant difference was noted between group 2 and group 3 ( P > .05). However, the prevalence of specific IgE antibody to TDI-HSA conjugate was not significantly different between group 1 and group 2 (14% vs 7.7%, P > .05) or group 2 and group 3 (7.7% vs 0%, P > .05). There was no significant difference in prevalence of specific IgE or specific IgG according to the type of asthmatic response during the TDI BPT ( P > .05). Overall, statistically significant association was noted between the prevalence of specific IgE and IgG antibodies in 103 subjects ( P < .05), but no difference was noted within group 1 subjects only ( P > .05). Conclusion: These findings demonstrate that the presence of serum-specific IgG is closely related to TDI BPT results, and it may contribute to the development of TDI-induced asthma. (J Allergy Clin Immunol 1999;104:847-51.)

Specific IgG response to monomeric and polymeric diphenylmethane diisocyanate conjugates in subjects with respiratory reactions to isocyanates

Background: Isocyanates are a frequent cause of occupational asthma and can also induce hypersensitivity pneumonitis. Objectives: It is still unclear whether antibodies to diphenylmethane diisocyanate (MDI), which are elicited in some subjects with these conditions, are specific for this type of isocyanate. Moreover, preparation of conjugates to human serum albumin (HSA) with the polymeric formulation rather than monomeric MDI might result in improved detection of antibodies. Methods: We addressed these issues by testing the sera of 13 subjects with asthma (n = 12) and hypersensitivity pneumonitis (n = 1) induced by MDI (n = 4 or 5, see below) by comparing them with sera obtained from subjects with occupational asthma caused by toluene diisocyanate (TDI; n = 5) and hexamethylene diisocyanate (HDI; n = 2). Conjugate preparations were compared by using SDS-PAGE, absorbance spectral analysis, and isolectric focusing. Immunologic screening was done by ELISA. Results: Specific IgG antibodies that recognize MDI-HSA conjugates were detected in all but 1 of the MDI-exposed workers and could not be found in TDI-exposed and HDI-exposed workers. The levels of specific IgG antibodies were more elevated when tested against the HSA conjugates formed with polymeric MDI compared with the HSA conjugates formed with monomeric MDI. Conclusion: This study shows that specific IgG antibodies to MDI appear to be specific for MDI without cross-reactivity with TDI and HDI and higher by use of polymeric rather than monomeric MDI-HSA test antigens. (J Allergy Clin Immunol 1999;103:749-55.)

Risk factors for sensitisation and respiratory symptoms among workers exposed to acid anhydrides: a cohort study.

OBJECTIVES: To examine the relation between exposure to acid anhydrides and the risk of developing immediate skin prick test responses to acid anhydride human serum albumin (AA-HSA) conjugates or work...

Monoclonal antibody monospecific to glycine for brain immunocytochemistry

We have developed mouse monoclonal antibodies (AGLY-1–8, all IgG 1 subisotype mAbs) against glycine (Gly) conjugated to bovine serum albumin using glutaraldehyde (GA)–NaBH 4. Among these, AGLY-4 mAb was found to be the most useful for Gly immunocytochemistry (ICC) in functions of specificity and sensitivity without non-specific immunobinding. AGLY-4 was demonstrated to be monospecific to Gly by an enzyme-linked immunosorbent assay (ELISA) binding test, and not reactive to any of the other amino acids and peptides tested. Using this antibody, indirect immunoperoxidase staining was observed in different regions of the rat brain fixed with GA in combination with borohydride reduction. In contrast, immunoreactivity was quite low in tissues fixed only with GA. Absorption controls indicated that the immunostaining could be completely inhibited by 5 μg/ml of Gly–human serum albumin (HSA) conjugate prepared using GA and NaBH 4, which was consistent with the results of an ELISA inhibition test. No cross-reaction occurred with other GA-conjugated amino acids. Dense ICC staining was observed in the rat neurons related to the auditory and vestibular centers, and modest immunostaining was seen in all the structures of the cerebellar cortex except for the Golgi cells which were strongly stained. These results were in complete agreement with the previous methods using polyclonal anti-Gly serum. Also, a new finding was that staining was noticed in certain cells widely distributed in the different brain regions. These results strongly suggest that the monoclonal antibody has a potential for elucidating the precise distribution of Gly-containing cells.

T-lymphocyte responses to plicatic acid–human serum albumin conjugate in occupational asthma caused by western red cedar

Background: T cells are known to play a major role in the pathogenesis of atopic allergic asthma, but it is less clear whether they are involved in occupational asthma caused by low molecular weight chemicals such as plicatic acid. Objectives: We sought to determine whether peripheral blood T cells from patients with western red cedar asthma (WRCA) recognize plicatic acid (PA) conjugated to human serum albumin (HSA) as judged by proliferation or cytokine production and to analyze the response to PA inhalation with flow cytometry. Results: Significant proliferative responses to PA-HSA were observed in eight of 33 patients with WRCA, none of 10 exposed nonasthmatic cedar workers, and one of 18 nonasthmatic control subjects. Two of 25 patients with WRCA also showed proliferative responses to unconjugated PA. All the WRCA responders were either currently exposed to cedar or had ceased exposure within the preceding 2 years. None of the four patients receiving oral steroids responded, but inhaled steroids did not seem to influence responsiveness. No correlations were found between the maximum stimulation response and any of the current FEV 1 values, the current PC 20 methacholine values, or the magnitude of the late asthmatic response to PA. Peripheral blood T-cell subset proportions and their degree of activation were similar in patients with WRCA and exposed control subjects. There was no change in T-cell phenotypes or their activation markers after PA inhalation challenge. In vitro, PA-HSA stimulation did not affect subset ratios but led to release of small amounts of IL-5 and IFN-γ, with no detectable increase in IL-4. Conclusions: PA-HSA–specific T lymphocytes seem to be present in small numbers in the peripheral blood of patients with WRCA and may respond to antigenic exposure by producing IFN-γ and IL-5. However, the proportion of responding cells would appear to be lower than in comparable studies of atopic asthma. (J Allergy Clin Immunol 1998;101:841-7.)

Targeting of Naproxen Covalently Linked to HSA to Sinusoidal Cell Types of the Liver

The kinetic behaviour of a naproxen human serum albumin conjugate (Nap23-HSA) was investigated in rats and in isolated perfused rat livers (IPRL), as compared to its active metabolite naproxen-lysine (Nap-lysine) and free naproxen. Through covalently linking the antiinflammatory drug naproxen to HSA, this drug can be selectively delivered to non parenchymal cells of the liver. Liver endothelial and Kupffer cells play an important role in the pathogenesis of inflammatory liver diseases. Targeting naproxen to these cells might increase its efficacy and reduce the side effects.The altered kinetic properties of Nap23-HSA, after i.v. injection of 22 mg.kg−1, as compared to an equimolar amount of the uncoupled drug, were demonstrated in vivo by a decrease in the steady state volume of distribution (41 ± 5 vs. 134 ± 19 ml.kg−1), a decrease in its clearance (0.48 ± 0.05 vs. 0.63 ± 0.1 ml.min−1.kg−1), a shorter plasma half life (60 ± 11 vs. 152 ± 44 min) and a sustained biliary excretion.Liver targeting of Nap23-HSA was clearly demonstrated: drug content of the liver 180 min after injection was about 30 times higher for Nap23-HSA as compared to naproxen itself. The IPRL experiments showed that the Vmax of hepatic removal of the conjugate was 40 μg.min−1.g liver−1. With doses below receptor saturation a rapid removal of the conjugate (t1/2 = 6 min) from the perfusion medium was found.In conclusion, this study demonstrates the saturable uptake of Nap23-HSA and its lysosomal degradation in both in vivo and IPRL experiments. Covalently linked naproxen is released as Nap-lysine. This active metabolite accumulates in Kupffer and endothelial cells in which it reaches therapeutic concentrations. Release from these cells leads to rapid uptake by hepatocytes and carrier mediated excretion into bile. Levels of Nap-lysine in bile and plasma reflect the slowest step in its generation: the proteolytic release in endothelial and Kupffer cells.

Risk factors for sensitisation to methyltetrahydrophthalic anhydride.

OBJECTIVES: To examine an association between specific IgE to methyltetrahydrophthalic anhydride (MTHPA) and exposure time, atopic history, smoking habits, and total IgE concentrations. METHODS: A cross sectional survey was carried...

Structural Determination of the Conjugate of Human Serum Albumin with a Mitomycin C Derivative, KW-2149, by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

A new mitomycin C derivative, KW-2149, is known to form a covalent conjugate with human serum albumin (HSA). This conjugate exhibits 1/20 of the anticellular activity of unconjugated KW-2149. Structural studies of this conjugate were carried out using a combination of enzymatic digestion, high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The tryptic peptide T5 (residues 21-41) was the only peptide found to be modified by KW-2149 moieties, the [(gamma-L-glutamylamino)ethyl]thio group or the (2-aminoethyl)thio group, through a disulfide bond. Although the latter peptide lost its mitomycin C moiety in the course of tryptic digestion, these data strongly suggest that KW-2149 was bound to Cys-34, the only free cysteine on HSA.

Use of a Heterologous Bridge Coating Antigen for the Immunoassay of 3-Acetyldeoxynivalenol in Barley

The study describes procedures that were used to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA) for the quantitation of a trichothecene mycotoxin, 3-acetyldeoxynivalenol (3-AcDON), in barley. Polyclonal antibodies were produced in rabbits immunized with a conjugate of 3-AcDON and human serum albumin linked by an ester linkage (hemisuccinate bridge). High anti-3-AcDON titers were obtained after multiple immunizations. However, only a negligible degree of inhibition was obtained with 3-AcDON in a competitive ELISA when the coating conjugate contained the same ester linkage group (hemiglutarate bridge) as the immunogen. The use of a conjugate containing a heterologous ether linkage (O-methylcarboxyl bridge) compared to that of the immunogen yielded an inhibition curve for 3-AcDON that was highly sensitive (IC50 = 0.21 ng/ml) with essentially no interference from the bridging group. This conjugate was synthesized using iodoacetate and 1,1′-carbonyldiimidazole chemistries. The assay showed low cross-reactivity with other trichothecenes including several analogs of deoxynivalenol (DON) with the exception of acetylated DON. The ELISA developed on the basis of this new conjugate was able to detect low concentrations of 3-AcDON (16 ppb) in spiked barley without any cleanup treatments.

Investigation of the mechanism of alcohol-induced bronchial asthma

BACKGROUND: Many Japanese patients with asthma experience episodes or exacerbation of asthma after alcohol consumption. This phenomenon is not seen in Caucasians and is specific to Asians. This has been thought to be attributable to a difference in alcohol metabolism, in particular the metabolism of acetaldehyde, between Asians and Caucasians. METHODS: An oral ethanol challenge test, a leukocyte histamine release test, and an ELISA for detection of IgE specific to acetaldehyde–human serum albumin conjugate were carried out in 42 adults with bronchial asthma and nine healthy adults.> RESULTS: Fifty-five percent of the patients with asthma responded to the ethanol challenge and showed a 20% or greater reduction in forced expiratory volume in 1 second. Blood acetaldehyde and plasma histamine levels were significantly higher in responders than in nonresponders. The leukocyte histamine release test revealed no ethanol-induced histamine release. Acetaldehyde, on the other hand, was found to induce histamine release in a volume-dependent manner. The histamine release was significantly higher in the asthma group (both responders and nonresponders) than in the healthy control group. ELISA did not detect any IgE specific to acetaldehyde–human serum albumin conjugate. CONCLUSION: Alcohol-induced bronchial asthma seems to develop as follows. Alcohol elevates blood acetaldehyde levels, which leads to degranulation of mast cells (or basophils). The resultant release of chemical mediators, such as histamine, induces asthma. (J A LLERGY C LIN I MMUNOL 1996;97:74-84.)

Active immunization of post-pubertal heifers against prostaglandin F 2α to suppress oestrous behaviour: effect of adjuvant and dose of immunogen

Two experiments were performed to develop an effective prostaglandin F 2α immunization protocol to suppress oestrous behaviour in beef heifers. In Experiment 1, a 3 × 2 factorial plan ( n=4–5 per treatment) was used to test three doses (3.3, 10 and 30 mg) of a prostaglandin F 2α- human serum albumin (PGF-HSA) conjugate as the immunogen and two adjuvants, GNE (proprietary product; Intervet, The Netherlands) and diethylaminoethyl (DEAE)-dextran. Heifers ( n=5) in a control group were untreated. Booster immunizations were given on Days 42 and 145 after the primary immunization (Day 0) and data collection for statistical purposes ended on Day 297. After Day 42 the incidence of oestrous behaviour was: (1) greater ( P < 0.05) for control than immunized heifers (4.3 and 2.2, respectively), (2) greater ( P < 0.05) for heifers immunized using GNE than for heifers immunized using DEAE-dextran (2.6 and 1.9, respectively), and (3) greater for heifers immunized with 30 mg of immunogen than for those immunized with either 3.3 or 10 mg (3.1, 1.7 and 1.9, respectively). Suppression of oestrous behaviour was accompanied by formation of a persistent corpus luteum (CL). Persistent CL were formed in ten of the 28 immunized heifers and the mean (± standard error of the mean) duration of persistence was 397 ± 85 days. In Experiment 2, a 2 × 2 factorial plan ( n=6–7 per treatment) was used to test two doses (1 and 10 mg) of the PGF-HSA conjugate as the immunogen and two adjuvants, non-ulcerative Freund's adjuvant (NUFA) and DEAE-dextran. A control group was untreated ( n=6). Booster immunization was given on Day 183 after the primary immunization (Day 0) and the experiment finished on Day 384. Antibody titres were higher ( P < 0.05) in NUFA-treated heifers than in DEAE-dextran-treated (1 mg) heifers in the 183- to 283-day period. After Day 183, oestrous behaviour was suppressed in 26 out of the 27 immunized heifers. Persistent CL were maintained for longer ( P < 0.05) in NUFA-treated heifers (245 days) than in DEAE-dextran-treated heifers (166 days) but there was no difference due to dose of immunogen (208 and 203 days, 1 and 10 mg, respectively). It is concluded that immunization against PGF-HSA results in suppression of oestrous behaviour in heifers due to prolongation of the life-span of the CL; however, efficacy of response is dependent on the immunization regime used.

Hypersensitivity pneumonitis (extrinsic allergic alveolitis) induced by isocyanates

Background: Chemical-induced hypersensitivity pneumonitis has been so far rarely described. The purpose of this study was to find out whether hypersensitivity pneumonitis is a common disorder in isocyanate workers. Methods: Company physicians' case histories of 1780 isocyanate workers were evaluated. In 16 subjects suspected of having isocyanate-induced hypersensitivity pneumonitis, chest x-ray films were made; levels of IgE and IgG antibodies to isocyanate–human serum albumin were estimated; conjugates and isocyanate challenge tests, bronchoalveolar lavage fluid analyses, and/or lung histologic investigations were performed. Results: Each of the 14 study patients who had hypersensitivity pneumonitis had work-related dyspnea and fever occurring several hours after the start of work with isocyanates. Typical clinical findings were the reduction of lung diffusing capacity ( n = 10), reticular or nodular lung patterns in the x-ray film ( n = 9), and serum IgG antibodies specific to isocyanate–human serum albumin conjugates ( n = 10). Restrictive ventilation patterns in the inhalation challenge tests ( n = 5), lymphocytic and/or neutrophilic alveolitis seen in bronchoalveolar lavage fluid analyses ( n = 7), and lymphohistocytic patterns mostly associated with mild fibrosis in lung histology ( n = 5) confirmed the diagnosis. Conclusion: Occupational exposure to isocyanate vapors and aerosols induces typical hypersensitivity pneumonitis in at least 1% of the isocyanate workers with symptoms. Diphenylmethan diisocyanate was found to be the main cause of this disorder. (J A LLERGY C LIN I MMUNOL 1995;95:1004-10.)