Clara cell secretory protein deficiency alters clara cell secretory apparatus and the protein composition of airway lining fluid

Clara cells represent the predominant secretory cell within distal conducting airways of mammals and exhibit functional alterations with chronic lung disease. We previously demonstrated that Clara cell secretory protein (CCSP) deficiency results in enhanced susceptibility to environmental agents. The present study was undertaken to define changes in Clara cell secretory function associated with CCSP deficiency in knockout mice. Comparative morphometry of Clara cell ultrastructure revealed dramatic alterations in secretory apparatus between wild-type (WT) and CCSP knockout (CCSP-/-) mice. Secretory granules, which occupy greater than 2% of Clara cell cytoplasmic volume in WT mice, were completely absent among Clara cells of CCSP-/- mice. Moreover, Clara cells of CCSP-/- mice exhibited a > 95% reduction in rough endoplasmic reticulum and alterations to Golgi apparatus, relative to WT controls. Ultrastructural perturbations to Clara cells were associated with altered protein composition of airway lining fluid as revealed by two-dimensional gel analysis of bronchoalveolar lavage proteins, but were not associated with altered abundance or secretion of CC26, another Clara cell secretory protein. We conclude that CCSP is required for the appearance of Clara cell secretory granules and that functional changes to Clara cells that result from CCSP deficiency lead to alterations in the composition of epithelial lining fluid.     

Double mutation (A171T and D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening the the United States. Mutations in brief no. 128. Online

Biotinidase deficiency is inherited as an antosomal recessive trait that, unless treated with pharmacologic doses of biotin, can result in neurologic and cutaneous symptoms. We have identified two new mutations in exon D of the biotinidase gene of children with profound biotinidase deficiency ascertained by newborn screening. Transition 511G->A near the 5' end of exon D results in a substitution of threonine for alanine 171 (A171T) and transversion 1330G->C occurs close to the 3' end of exon D causing a substitution of histidine for aspartic acid 444 (D444H). The D444H mutation was detected in four individuals from our normal population whose mean serum biotinidase activity is 5.25 nmol/min/ml, which is significantly lower than the mean normal activity (7.1 nmol/min/ml). We calculated that this mutation causes a 52% loss of activity in the aberrant enzyme. Twenty-three individuals with the D444H mutation were found by allele specific oligonucleotide analysis of DNA from 296 randomly-selected, anonymous dried-blood spots. We estimate the frequency of this allele in the general population to be 0.039. In contrast, no individuals in 376 have the A171T mutation. Fourteen children (eleven probands and three siblings) out of the 31 enzyme-deficient children have both the A171T and D444H mutations. Both mutations are inherited from a single parent as a double mutation allele. The nine families in which this allele was identified are of mostly European ancestry, although the mutation cannot be attributed to a specific nationality or ethnic group. The serum of a child who is homozygous for the double mutation allele has very little CRM and the aberrant enzyme has very low biotinylhydrolase activity and no botinyl-transferase activity. This double mutation allele (A171T and D444H) is a common cause of profound biotinidase deficience in children ascertained by newborn screening in the United States.     

Reciprocal translocation mosaicism in man

We report on an8-month-old girl with a de novo 5q/6q autosomal translocation with loss of the distal part of the long arm of chromosome 6 (6q23.3→6qter); clinical manifestations are peculiar craniofacial stigmata, truncal obesity and persisting hypotonia. The similarity with a previously reported patient with 6q interstitial deletion is discussed.     

The Genée-Wiedemann syndrome,an acrofacial dysostosis—further observation

We report on a consanguineous Brazilian couple whose 2 children had tibial aplasia-ectrodactyly. Femoral bifurcation was present in one of the affected children. The relationship of tibial aplasia-ectrodactyly to the Gollop-Wolfgang complex is discussed. Clinical and genetic aspects of the conditions involving tibial aplasia and femoral bifurcation are discussed.     

Apoptosis of ileal Peyer's patch B cells is increased by glucocorticoids or anti-immunoglobulin antibodies

The ileal Peyer's patch (PP) in sheep plays a central role in the development and production of B cells. Associated with a tremendous amount of B cell proliferation in this site is the extensive diversification of the Ig repertoire by somatic hypermutation. Very few (<5%) of the B cells produced in the ileal PP differentiate and emigrate; instead, the vast majority of these cells soon die, and we have previously shown that death is associated with apoptosis. When placed in culture, ileal PP B cells die rapidly by apoptosis, such that after 24h, 60 ± 1 % of DNA is fragmented. Here, we show that the extent of this spontaneous B cell apoptosis in culture, as quantitated by DNA fragmentation, was significantly increased in a dose-dependent manner by the glucocorticoids hydrocortisone or dexamethasone. Furthermore, treatment of lambs with 2–2.5 mg/kg of dexamethasone resulted in a marked increase in the number of apoptotic cells in the ileal PP and an increase in ileal PP B cell DNA fragmentation to 20 ± 6%, compared with 2.4 ± 0.1 % in untreated lambs. Anti-immunoglobulin (Ig) antibodies also increased the extent of DNA fragmentation in cultured ileal PP B cells. After 24 or 48 h of culture with anti-Ig (PIg47A), DNA fragmentation was 74 ± 2 % and 75 ± 3 %, respectively. Ileal PP B cells are rescued from apoptosis by agents that activate protein kinase C and increase cytosolic Ca²⁺, and here we show that this treatment also results in apoptotic rescue in the presence of dexamethasone or anti-Ig. We speculate that the apoptosis of ileal PP B cells in situ may be modulated by glucocorticoids and by the cross-linking of surface Ig. Apoptosis, induced by a signal through surface Ig, may be an important mechanism in the deletion of self-reactive B cells during the expansion of the Ig repertoire in the ileal PP.     

Urofacial (Ochoa) syndrome

Between 1965 and 1986 we saw 36 children with enuresis and urinary tract infection in association with “inversion” of facial expression when laughing. Urologic work-up of these patients disclosed characteristic findings of mild neuropathic bladder in all cases, with severe urinary tract damage in most of them. The clear association of distortion in facial expression and neuropathic bladder with resultant damage to the genitourinary tract should prompt urological evaluation of individuals with “inversion” of facial expression. About two thirds of the patients also had moderate to severe constipation. We suggest the term urofacial syndrome for this disorder. The occurrence of the disorder in multiple sibs, normal parents, increased parental consanguinity, and equal sex ratio indicate autosomal recessive inheritance.     

Studies of the transport of polyclonal IgA antibody from blood to bile in rats.

Bile or thoracic duct lymph, collected from rats 7-9 days after suspensions of B. abortus, S. typhi or SRBC had been injected into the Peyer''s patches, contained high titres of specific agglutinins. Samples of these fluids were injected i.v. into unimmunized, syngeneic recipients and the partitioning between blood and bile of the injected antibodies was studied and found to depend on the source and class of the antibody. IgA antibodies from lymph plasma disappeared rapidly from the recipients'' blood and half of the dose was recovered in the bile within 2 h of its injection. IgA antibodies which had been collected from bile and so had previously traversed the liver and acquired secretory component, appeared in the recipients'' bile much less rapidly so that less than half of the dose entered the bile over a period of 40 h. Passively administered IgG antibodies did not enter the recipients'' bile to any significant extent and specific haemolysins never appeared in the bile after either passive or active immunization.     

Terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction

Cellular mechanisms contributing to renewal of terminal bronchioles remain poorly defined. Our previous studies identified pollutant-resistant Clara cell secretory protein (CCSP)-expressing stem cells that localize to the neuroepithelial body (NEB) and contribute to renewal of the proximal bronchiolar epithelium. However, activation of NEB-associated stem cells is unlikely to contribute to renewal of terminal bronchiolar epithelium because of the paucity of NEBs at this location. Goals of this study were to determine the location and properties of cells contributing to renewal of terminal bronchioles after Clara cell depletion. Pollutant-resistant CCSP-expressing cells were identified that localized to the bronchoalveolar duct junction (BADJ) and contribute to restoration of a phenotypically diverse epithelium. CCSP-expressing cells comprise the predominant proliferative population in initial terminal bronchiolar repair and include a population of label-retaining cells suggesting that they maintain characteristics of a stem cell population. Furthermore, immunohistochemical co-localization studies involving CCSP and the NEB-specific marker calcitonin gene-related peptide indicate that BADJ-associated CCSP-expressing stem cells function independently of NEB microenvironments. These studies identify a BADJ-associated, NEB-independent, CCSP-expressing stem cell population in terminal bronchioles and support the notion that regiospecific stem cell niches function to maintain epithelial diversity after injury.     

Mass spectrometric identification of mtb81, a novel serological marker for tuberculosis

We have used serological proteome analysis in conjunction with tandem mass spectrometry to identify and sequence a novel protein, Mtb81, which may be useful for the diagnosis of tuberculosis (TB), especially for patients coinfected with human immunodeficiency virus (HIV). Recombinant Mtb81 was tested by an enzyme-linked immunosorbent assay to detect antibodies in 25 of 27 TB patients (92%) seropositive for HIV as well as in 38 of 67 individuals (57%) who were TB positive alone. No reactivity was observed in 11 of 11 individuals (100%) who were HIV seropositive alone. In addition, neither sera from purified protein derivative (PPD)-negative (0 of 29) nor sera from healthy (0 of 45) blood donors tested positive with Mtb81. Only 2 of 57 of PPD-positive individuals tested positive with Mtb81. Sera from individuals with smear-positive TB and seropositive for HIV but who had tested negative for TB in the 38-kDa antigen immunodiagnostic assay were also tested for reactivity against Mtb81, as were sera from individuals with lung cancer and pneumonia. Mtb81 reacted with 26 of 37 HIV(+) TB(+) sera (70%) in this group, compared to 2 of 37 (5%) that reacted with the 38-kDa antigen. Together, these results demonstrate that Mtb81 may be a promising complementary antigen for the serodiagnosis of TB.