Trypsin activity in stool and duodenal aspirate of neonates for differential diagnosis of intestinal obstruction.

The diagnosis of a non-surgical cause of delayed passage of meconium in a neonate may be challenging to the pediatric surgeon. The usefulness of determining trypsin activity (TA) in stool and duodenal aspirate for the diagnosis of cystic fibrosis (CF) and the existence of an entity called transient deficiency of trypsin (TTD) was assessed in 49 neonates over a 14-month period. TA was determined by a gelatin liquefaction technique. An absence of TA was considered if gelatin liquefaction was seen at a dilution of less than 1:100. Neonates with negative activity in both samples were started on supplementary pancreatic enzymes (pancreatin) for 1 month. TA was retested in the stool after stopping pancreatin for 1 week. Neonates with a return of TA were considered to have TTD; those who showed persistent TA deficiency at re-evaluation were investigated for CF by sweat iontophoresis. Twenty-one neonates had negative TA (in 1 only stool was tested); 13 could be re-evaluated (4 died, 4 were lost to follow-up). Nine were found to have TTD as a stool or duodenal sample showed the presence of TA; 3 of 4 patients with persistent negative TA were confirmed to have CF. One patient with normal sweat chloride is awaiting genetic studies. Determination of TA by gelatin liquefaction is a simple, rapid, inexpensive, and reliable (sensitivity 100%, negative predictive value 100%) means to differentiate non-surgical causes of intestinal obstruction such as neonatal and postoperative TTD and CF.

Relationship between intracellular calcium and airway reactivity in guinea pigs.

The present study was carried out to examine the relationship between intracellular free calcium ion concentrations and its regulatory enzymes, sodium potassium adenosine triphosphatase (Na(+),K(+)-ATPase) and calcium adenosine triphosphatase (Ca(2+)-ATPase), with airway reactivity to inhaled histamine in guinea pigs. Forty-nine guinea pigs were included in this study. Of these, 34 animals responded to histamine bronchoprovocation challenge in vivo with a greater than 35% fall in specific airways conductance and were labeled as "reactive," and the remaining 15 were "nonreactive." The dose of histamine producing a 35% fall in specific airways conductance was labeled as ED(35) SGaw. The animals were then sacrificed, and the following biochemical measurements were carried out: intracellular free calcium ion concentrations [Ca(2+)](i) in leukocytes and isolated tracheal smooth muscle cells, activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase in tracheal homogenate, and plasma levels of lysophosphatidylcholine (LPC). Reactive guinea pigs showed significantly higher [Ca(2+)](i) and Na(+),K(+)-ATPase and Ca(2+)-ATPase activities. Airway reactivity (ED(35) SGaw) had significant negative correlation with [Ca(2+)](i), with activities of each of the ATPases and with plasma lysophosphatidylcholine. It is concluded that the level of [Ca(2+)](i) is an important determinant of airway reactivity. Intracellular calcium levels modulate airway response to histamine with higher levels being associated with greater reactivity.

Fusarium solani major allergen peptide IV-1 binds IgE but does not release histamine.

BACKGROUND: Fusarium solani (FS) is an important allergen source afflicting 4% of the nasobronchial allergy patients. Fus s I3596*, a 65 kDa major glycoprotein allergen of FS reacts with 95% fungus sensitive patients. OBJECTIVES: To purify and characterize a potent peptide from Fus s I3596* which may be useful for therapeutic purposes. METHODS: The 65 kDa protein was sequentially cleaved with trypsin and cyanogen bromide (CNBr). The cleaved products were purified on reverse phase high performance liquid chromatography (rpHPLC) column and functionally characterized by in vitro and in vivo methods for its IgE binding and histamine release. RESULTS: The protein on cleavage showed 11 peaks (I to XI). Of these, peaks I, III, IV and V were highly allergenic as determined by IgE ELISA. These peaks were further purified and peptide IV-1 was most potent in comparison to other peptides by ELISA-inhibition. This peptide showed IgE binding but could not evoke intradermal response in Fusarium-sensitive patients. Heparinized blood challenged with peptide IV-1 does not release histamine. Preincubation of heparinized blood with peptide IV-1 and challenging with crude extract blocked histamine release in a dose dependent manner. CONCLUSION: Peptide IV-1 binds to IgE but does not release histamine, demonstrating its potential use in therapy of Fusarium-allergic patients.

Purification and partial characterization ofa 67-kD cross-react ive allergen from Imperata cylindrica pollen extract.

BACKGROUND: Grass pollens are known to induce type I allergic reactions in a large number of genetically predisposed individuals. Earlier studies have recognized Imperata cylindrica (Ic) pollen as an important source of aeroallergen which contained 7 IgE binding proteins in the MW range of 85-16 kD. OBJECTIVES: To isolate, purify and characterize a cross-reactive allergenic protein from Ic pollen extract for diagnosis and therapy of grass pollen allergy. METHODOLOGY: Ic pollen extract was fractionated using DEAE Sephadex A-50, Sephadex G-200 and Mono Q column. Allergenic activity of the fractions was checked by ELISA, skin tests, ELISA inhibition and immunoblot using sera of Ic-sensitive patients. A 67-kD protein was purified to homogeneity from Ic-VIII. The allergenic determinants of this protein were identified by SDS-PAGE and immunoblot after CNBr treatment. RESULTS: Among Ic fractions, Ic-VIII was highly potent by ELISA, skin tests and showed cross-reactivity with 4 other tropical grasses by immunoblot and ELISA inhibition. The subfraction Ic-VIIIe1 of Ic-VIII showed a band at 67 kD on SDS-PAGE. On CNBr treatment, it gave 7 peptides, 3 of which were found to be allergenic. CONCLUSION: A 67-kD protein (Ic-VIIIe1) was isolated, purified to homogeneity and partially characterized. It showed cross-reactivity with tropical grasses tested and contained at least three allergenic determinants.

Distribution of beta-thalassemia mutations in the Indian population referred to a diagnostic center.

Hemoglobinopathies are the most commonly inherited genetic disorders in India. Population screening has identified certain communities in India with high risk of beta-thalassemia, the prevalence of carrier status in some being as high as 17%. Over a period of 6 years we have conducted DNA analysis on 1,233 carriers of 23 beta-thalassemia mutations and Hb E, using the amplification refractory mutation system. The studies included analyses of five common mutations for Asian Indians, namely IVS-I-5 (G-->C), 619 bp deletion, IVS-I-1 (G-->T), and the frameshifts at codons 8/9 (+G) and 41/42 (-TTCT). The occurrence of these was seen in 1,066 (86.45%) of the carriers referred to us, the percentage of mutations varying between 5.03-42.58%. We found codon 15 (TGG-->TAG) in 47 (3.81%) samples which was also considered a common mutation in the Indian population. Rare beta-thalassemia mutations were found in 87 (7.06%) individuals. We have designed five new primers and modified four primers for nine rare mutations. These were seen in nine (0.73%) samples. The beta-thalassemia anomaly in 33 (2.68%) carriers remained uncharacterized. State-wide and community-wide distribution patterns of mutations indicated that IVS-I-5 (G-->C) is the most common beta-thalassemia allele in the Indian population. Sindhis settled in Gujrat, and Maharashtra and Lohanas from Gujrat showed a prevalence of the 619 bp deletion mutation in 49.2 and 45.5% carriers, respectively. High frequency of the IVS-I-1 (G-->T) mutation was also found in Sindhis (25.5%), Punjabi Hindus (34.7%), and Lohanas (31.2%). These studies of mutation patterns in different communities have helped us in the quick identification of beta-thalassemia mutations for prenatal diagnosis.

Development of allergen-coated paper discs for allergy diagnosis by ELISA.

In vitro testing is useful for detecting pollen and fungal-allergen sensitivities in nasobronchial-allergy patients. However, allergen-coated discs or solid-phases supplied in kits do not contain all the relevant extracts. The present study was aimed to develop a safe and credible procedure of allergen coating on paper discs for allergy diagnosis by ELISA. Paper discs were coated with indigenous allergens using poly(vinyl alcohol) and glutaraldehyde. ELISAs were carried out to standardize the allergen-coated discs using skin-test-positive-patients' sera. The cut-off value (A) for ELISA was worked out by analysing normal human sera (NHS) and is based on the mean+/-2 S.D. for the NHS value. The allergen-coated discs, i.e. Centre for Biochemical Technology (CBT) discs, demonstrated excellent comparison with plate-binding method and Pharmacia discs [Prosopis juliflora (mesquite), critical value 0.879 at 5% probability+/-0.599, n=11, is significant]. Inter- and Intra-assay coefficients of variations ranged from 2.5-14.05% to 4. 8-11.5% with different allergens. Results of skin tests and ELISA with CBT discs showed 60-70% correlation.

Prenatal diagnosis of beta-thalassaemia and other haemoglobinopathies in India.

This paper reports prenatal diagnosis of 787 fetuses of beta-thalassaemia and other haemoglobinopathies in Indian high-risk communities. DNA based diagnosis was offered in the first, as well as the second trimester, in 489 pregnancies (with five twins) on fetal tissues such as chorionic villus (CV) and amniocytes using the amplification refractory mutation system (ARMS) and restriction fragment length polymorphism (RFLP) techniques. Two hundred and ninety-two women (with one twin), who either presented late in the second trimester or whose DNA diagnosis was not informative, were offered prenatal diagnosis using globin chain synthesis (GCS) on fetal blood cells. Maternal contamination of fetal DNA was ruled out by variable number tandem repeat (VNTR) analysis using sites in four different genes (Apo-B, D1S-80, Ig-JH and Ha-ras), while contamination of fetal blood was checked by a particle size distribution channelyzer. Using both techniques we were able to offer complete diagnosis in 99.8% cases. Out of 494 fetuses tested by DNA analysis, 135 were found to be normal, 201 were carriers, whereas 146 were affected. Out of 293 fetuses analysed by GCS, 215 were unaffected and 71 were affected. In this study, both fetuses were tested in twin pregnancies, of which three required selective termination of one fetus. Because of social, religious taboos and family influences, genetic counselling was found to be an important guideline for couples selecting options for prenatal diagnosis. Our experience suggests that because of late presentation by many couples to the diagnostic centres, in developing countries like India, both the techniques of DNA analysis and GCS should be made available at major referral centres for maximum benefit to couples.

Characterization of cogon grass (Imperata cylindrica) pollen extract and preliminary analysis of grass group 1, 4 and 5 homologues using monoclonal antibodies to Phleum pratense.

BACKGROUND: Previous studies have established the role of Imperata cylindrica (Ic) pollen in type I allergic disorders. However, no systematic information is available on the allergen composition of Ic pollen extract. OBJECTIVES: To characterize the IgE-binding proteins of Ic pollen extract and to detect the presence of grass group 1, 4 and 5 allergen homologues, if any. METHODS: Pollen extract of Ic was analyzed by in vivo and in vitro procedures such as intradermal tests (ID), enzyme-linked immunosorbent assay (ELISA), ELISA-inhibition, thin-layer isoelectric focusing (TLIEF), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Dot blot assay was carried out to check the presence of well-known group 1, 4, and 5 allergen homologues in Ic pollen extract. RESULTS: Out of 303 respiratory allergies patients skin-tested, 27 showed sensitivity to Ic pollen extract. Specific IgE levels were elevated in all 15 serum samples tested. The extract prepared for this study was found to be highly potent since it required only 400 ng of homologous proteins for 50% inhibition of binding in ELISA inhibition assays. TLIEF of Ic pollen extract showed 44 silver-stained bands (pI 3.5-7.0) while SDS-PAGE resolved it into 24 Coomassie-Brilliant-Blue-stained bands (MW 100-10 kD). Immunoblotting with individual patient sera recognized 7 major IgE-binding bands (MW 85, 62, 57, 43, 40, 28 and 16 kD) in Ic pollen extract. A panel of monoclonal antibodies, specific to group 1, 4 and 5 allergens from Phleum pratense pollen extract identified group 5 and group 4 homologues in Ic pollen extract. CONCLUSION: Ic pollen extract was characterized for the protein profile by TLIEF and SDS-PAGE. IgE reactivity was determined by ELISA and immunoblot. Monoclonal antibodies to group 5 and group 4 allergens reacted weakly showing that this pollen contains group 5 and group 4 homologous allergens.

Role of liposomes in selective proliferation of splenic lymphocytes.

The effects of free and encapsulated allergens of Artemisia scoparia pollen on lymphocyte proliferation and immunoglobulin production in BALB/c mice were investigated. Splenic lymphocytes from mice immunized with liposome entrapped allergen (LEA) elicited a marked proliferative response upon in vitro stimulation with both free and encapsulated allergen in comparison to mice immunized with free allergen (FA). The serum immunoglobulin profile of mice administered LEA revealed a predominance of IgG1 antibodies concomitant with an enhancement of IgG2a, IgG2b, IgG3 and IgM responses and suppression of IgE responses. However immunization with FA resulted in significant production of IgE responses and low levels of IgG antibodies. The differential ability of free and encapsulated allergens to selectively induce immunoglobulin isotypes suggests that different presentation and T cell differentiation pathways may be followed by FA and LEA in the immune system. Proliferation studies involving macrophage depletion demonstrated that macrophages play an obligatory role in the processing of LEA. Analysis of cytokine production in sera of immunized mice (FA/LEA) revealed that LEA induced significant IFN-gamma responses and lower IL-4 responses than mice immunized with FA. The results of the present study indicate that liposomes synergise the proliferation by the antigen incorporated in it and polarizes the response towards Th1 type of cytokine production. The immunoadjuvant and immunomodulation property of liposomes make it an efficient vehicle for effective immunotherapy.

Isolation and immunobiochemical characterization of a major allergen (65 kDa) from Fusarium equiseti.

Fusarium equiseti is one of the most important species in the class Deuteromycetes (Fungi Imperfecti). For proper diagnosis and immunotherapy, isolation and characterization of allergens of F. equiseti are necessary. In the present study, culture filtrate (CF) extract of F. equiseti was resolved into 35-37 bands on isoelectric focusing pI (3-9) and SDS-PAGE (mol. wt. 10-100 kDa). Most of them were glycoproteins, as identified by PAS staining. F. equiseti CF revealed 15 allergenic proteins on immunoblot with an allergic serum pool. It was fractionated into nine fractions (I-IX) on a Superose-12 column by FPLC. Fraction IV (65 kDa) and fraction VI (25 kDa) were found to be highly allergenic by IgE ELISA. A 65-kDa protein was observed as a major allergen because it was recognized by most of the patient sera on immunoblot. After elution from SDS-PAGE gel, it gave two bands of pI 7.4 and 6.0. Inhibition in IgE-binding components of F. equiseti CF with CF extracts of F. solani and F. moniliforme by immunoprint inhibition assay indicated the allergenicity shared between the extracts of Fusarium species. Data suggested that the 65-kDa is the major allergen in the Fusarium species and can be used for the treatment of allergic patients.

Liposome entrapped allergen reduces plasma histamine in sensitized mice.

Immunotherapy of allergic diseases is associated with problems of adverse systemic reactions. We have shown earlier that liposome entrapped allergen (LEA) is effective in inducing IgG response and restricting IgE response in immunized mice. This mode of treatment may be more effective and safer if it can prevent anaphylaxis. To determine this feature, mice were administered allergen preparations repeatedly and later challenged with the same allergen. Mice given liposomal preparation showed lower specific IgE response as compared to the mice given free allergen or alum adsorbed allergen of Artemisia scoparia. Specific IgG response was higher in mice immunized with LEA. The mice immunized with liposomal preparation survived whereas others injected with free allergen or alum adsorbed allergen died probably due to anaphylaxis. High levels of histamine were observed in mice injected with free allergen as compared to the mice injected LEA. The increase in plasma histamine level may be the cause of anaphylaxis during allergen challenge. In conclusion, LEA could be used as a safe and effective mode of immunotherapy for allergy diseases, since it reduces plasma histamine levels considerably thereby reducing the chances of anaphylaxis.

Polarized TH1 responses by liposome-entrapped allergen and its potential in immunotherapy of allergic disorders.

BACKGROUND: The conventional immunotherapy used for treating allergic individuals may at times lead to varying degrees of anaphylactic reaction. Liposomes have been proposed as a vehicle for safe and effective allergen-specific immunotherapy as multiple injections of liposome-entrapped allergen (LEA) have been shown to reduce specific IgE response and induce specific IgG response in BALB/c mice. OBJECTIVE AND METHODS: To elucidate the effect of LEA on polarization of T-cell responses, its effect on the relative production of TH1/TH2 type cytokines (namely IL-2, IL-4 and IFN-gamma by commercially available ELISA kits and immunoglobulin profile as measured by ELISA) were studied. Histamine release on challenge of immunized mice was measured to examine the efficacy of LEA in preventing anaphylactic reactions. RESULTS: Measurement of cytokine levels in serum and spleen cell culture supernatants of BALB/c mice injected repeatedly with either free allergen (FA) or LEA (three mice per group) indicate that LEA preferentially induces a TH1-type of response dominated by IFN-gamma and IL-2 production. Further, it was also shown that immunization of mice with FA or LEA and subsequent challenge with a large dose of the sensitizing allergen leads to fatal systemic reactions in 50-65% of the animals treated with FA, whereas no mortality was observed in mice injected with LEA. Analysis of IgG subclasses in sera of mice immunized with LEA revealed a sixfold higher production of IgG1 antibodies than mice immunized with FA. Serum IgG2a, IgG2b, IgG3 and IgM responses were also enhanced in the group of mice immunized with LEA in comparison with mice injected with FA. CONCLUSION: The results indicate that LEA confers protection against anaphylaxis to mice due to their ability to induce a high IFN-gamma:IL-4 ratio which may lead to decreased synthesis of IgE and reduced histamine release on challenge with FA.

Immunobiochemical characterization of Putranjiva roxburghii pollen extract and cross-reactivity with Ricinus communis.

Putranjiva roxburghii (PR) pollen has been found to be an important aeroallergen for type I hypersensitivity. In the present study, the IgE binding proteins of PR pollen have been characterized and compared with pollen allergens of Ricinus communis (RC) belonging to family Euphorbiaceae. On isoelectric focusing, PR pollen extract resolved into 35 bands (pI 3-9), whereas SDS-PAGE separated it into 18 protein components (MW 14-100 kD). Pooled patient's sera (ID +ve to PR) recognized 12 allergenic proteins in Putranjiva and five of them (MWs 92, 80, 55, 43 and 30 kD) showed immunologic reactivity to most of the sera samples tested individually by immunoblot. A number of shared allergenic proteins (MWs 92, 80, 66, 50, 43 and 14 kD) were observed between PR and RC pollen extracts on immunoblot using Putranjiva allergic serum pool. Inhibition in the binding for most of PR pollen allergenic proteins was obtained with higher concentration of RC extract than PR itself, depicting the presence of cross-reacting allergens in both. Putranjiva pollen extract was fractionated by a combination of DEAE Sephadex-A 50 and Sephadex-G 200 column chromatography. Periodate deglycosylation of western blotted PR extract and Put I fraction indicated the involvement of carbohydrate moieties in the allergenic activity. Of the two fractions from Put I (Ia and Ib), Put Ib was found to be the most allergenic protein by ELISA inhibition. Dot blot analysis with individual patients sera identified it as a major allergen of PR.

Ability of lymphokine-activated killer cells to lyse mycobacteria-infected cells.

Lymphokine-activated killer (LAK) cells were generated by interleukin-2 activation of peripheral blood lymphocytes obtained from lepromatous leprosy (LL) patients and healthy individuals. The ability of LAK cells to lyse targets (macrophages and T-24, a bladder carcinoma cell line) infected with mycobacteria (Mycobacterium leprae and mycobacterial strain ICRC) was assessed in a 51 chromium-release assay. It was observed that LAK cells generated from LL patients and healthy individuals could preferentially lyse M. leprae or ICRC-pulsed macrophages and T-24 cells, compared to non-pulsed targets. The ability of LAK cells to kill intracellular mycobacteria was demonstrated in colony forming assays. These results indicate a promising role for LAK cells in immunotherapy of leprosy.

Analysis of DNA ploidy and expression of tumour-associated antigens on human oral carcinomas xenografted in nude mice.

Human squamous cell carcinomas (SCC) of the oral cavity were successfully established as xenografts in nude mice. Tumours with higher malignancy scores and involvement of lymph nodes in patients were more readily accepted as xenografts in nude mice. The xenografted tumours were characterised with respect to morphology, histology, DNA index and expression of tumour-associated antigens (TAA). Flow cytometric analysis of cellular DNA content revealed that many of the xenografts retained the parent tumour DNA pattern while some of the xenografts showed progression to aneuploidy. All the xenografted tumours expressed TAA recognised by monoclonal antibody (MAb) 3F8E3. On Western blotting, MAb 3F8E3 recognised proteins of molecular weight 62-64 kDa on parent and xenografted tumours. In general, the xenografts reflect many of the characteristics of the tumours from which they were derived and may provide a useful model for investigating newer approaches of treatment and diagnosis.

Antisphingolipid antibodies in the sera of leprosy patients.

Earlier we reported the presence of significant levels of antigalactocerebroside (GalC) antibodies in the sera of leprosy patients. This study corroborates the above result and also gives evidence for the presence of antibodies to the nonpolar ceramide (Cer) moiety of GalC. AntiCer antibody titres were higher as compared to antiGalC antibodies in all categories of leprosy. The specificity of antibodies directed to the Cer moiety was confirmed using Lactosyl-BSA and neutralization assays. Statistically significant and positive correlations were observed between antiGalC and antiCer antibodies. Responsiveness factors were computed using natural logarithmic transformation of the variables.

Studies on shared antigenic/allergenic components among fungi.

Fungal allergens have been found to be one of the most prevalent aeroallergens in India. Knowledge of shared/unique components among different fungi is necessary for proper diagnosis and treatment of patients allergic to fungi. In the present study, crude extracts (CE) of 11 common fungi (Alternaria alternata, Aspergillus flavus, Asp. fumigatus, Asp. niger, Asp. tamarii, Asp. versicolor, Cladosporium herbarum, Curvularia lunata, Mucor hiemalis, Penicillium citrinum, and Fusarium solani) were characterized by isoelectric focusing (IEF), SDS-PAGE, and immunoblot. On IEF (pI 3-9), the number of protein bands was found to be greatest (46) in M. hiemalis extract. SDS-PAGE exhibited a varied number of bands, generally 18-40, with mol. mass ranging from 14 to 100 kDa. IgG-specific immunoprint using rabbit anti-F. solani CF antibodies demonstrated a mol. mass distribution of shared antigenic proteins of 14-100 kDa in most of the fungi. Shared allergenicity was observed in a number of allergenic proteins in fungal extracts with mol. mass ranging between 14 and 70 kDa on IgE-specific immunoblot using pooled sera of patients allergic to Fusarium. A 45-kDa protein was found to be common among these fungi on immunoblot with patients as well as with rabbit antibodies. F. solani CF extract contained more antigenic/allergenic proteins than F. solani CE. It was concluded that F. solani CF shared several antigenic/allergenic components with CE of other common fungi. This fact needs to be taken into account when fungal extracts are used in diagnosis and immunotherapy of allergic patients.

Immunobiochemical characterization of Brassica campestris pollen allergen.

Brassica campestris (BC), Eng. Mustard, is an important source of pollen allergen, responsible for type I hypersensitivity disorders. In the present study, BC pollen extract was characterized by TLIEF, SDS-PAGE and immunoprinting. The extract separated into 50 silver stained bands of pI 3-9 on isoelectric focusing whereas it resolved into 14 Coomassie blue stained protein bands of 14-100 kD on SDS-PAGE. Immunoblot analysis with individual patient sera detected four allergenic proteins of 90, 67, 60 and 14 kD. BC separated into 8 peaks (Bras 1-8) on DEAE Sephadex A-50 column. Bras 2 was found to be most potent by IgE specific ELISA, hence further fractionated on Sephadex G-200. A protein of 90 kD (Bras 2a) isolated by gel filtration was found to be most allergenic protein by ELISA inhibition. The findings shall be applicable in standardization of future batches of BC pollen extract to be used for allergy diagnosis and immunotherapy.