Analysis of Mycobacterium avium complex isolates from blood samples of AIDS patients by pulsed-field gel electrophoresis.

A molecular analysis of the first Mycobacterium avium complex (MAC) blood isolates from 177 patients from 10 Canadian cities revealed that each cluster of indistinguishable strains consisted of isolates from epidemiologically unrelated patients in the same city or region. This study supports the premise that acquisition of MAC from a common environmental source occasionally occurs.

Corneal ulceration in the elderly in Hyderabad, south India.

AIMS: To report demographic, microbiological, therapeutic, anatomical, and visual results of corneal ulceration in the elderly patients seen at a tertiary eye care centre in south India. METHODS: 102 consecutive cases of microbial keratitis in patients 65 years and older were studied. Inclusion criteria were: (i) presence of corneal stromal infiltrate upon slit lamp examination; and (ii) microbiological evaluation of corneal scrapings for suspected microbial keratitis. RESULTS: The principal predisposing factors identified in this study were ocular disease (38.2%), previous ocular surgery in the same eye (29.4%), trauma (17.6%), and severe systemic disease (16.7%). Contact lens wear was associated with only two cases (2.0%). 99 organisms were isolated in cultures of corneal scrapings from 74 (72.5%) of the 102 cases. Staphylococcus epidermidis (31.1%), filamentous fungi (25.7%), and Streptococcus pneumoniae (13.5%) were the most common isolates. 12 eyes (11.8%) required surgery, 15 (14.7%) eventually required evisceration, and nine (9.6%) of the 94 followed patients achieved an unaided vision of 20/60 or better at last follow up. CONCLUSIONS: This work represents the largest recent single centre study on (non-viral) microbial keratitis in the elderly, its management, and outcomes of therapy. While the predisposing factors differ from those of general population, the spectrum of microbes responsible for keratitis in the elderly appears to reflect the local microbial flora rather than a predilection for elderly patients. Delay in diagnosis and systemic conditions associated with advancing age probably contribute to poorer outcome from therapeutic measures.

Immune mediated 'HAART' attack during treatment for tuberculosis. Highly active antiretroviral therapy.

Highly active antiretroviral therapy (HAART) suppresses viral replication and improves immune function. However the inflammatory component of immune restoration can have clinically deleterious effects on previously asymptomatic infections. We report the development of acute respiratory failure in a patient after the institution of HAART, following 2 months of appropriate therapy for pulmonary tuberculosis. Necrotizing granulomas with acid-fast bacilli were found on lung biopsy, but cultures were negative for Mycobacterium tuberculosis and no other pathogens were isolated. Polymerase chain reaction of lung biopsy tissue for all mycobacterial species was positive only for M. tuberculosis. Rapid clinical improvement followed corticosteroid therapy. After initiating HAART, clinicians should be aware of the possibility of an inflammatory response to a previously quiescent tuberculous infection, even while on antituberculosis therapy.

Microbiologic spectrum and susceptibility of isolates: part I. Postoperative endophthalmitis. Endophthalmitis Research Group.

PURPOSE: To present the microbial spectrum and susceptibilities of isolates in postoperative endophthalmitis. METHOD: Isolates from 206 eyes of 206 patients who underwent vitrectomy for postoperative endophthalmitis were examined. RESULTS: One-hundred twelve (54.4%) of 206 vitreous samples were culture positive and 14 (12.5%) of 112 culture-positive cases were polymicrobial, yielding a total of 126 isolates. Isolates included 59 (46.8%) gram-positive cocci, eight (6.3%) gram-positive bacilli, 33 (26.2%) gram-negative organisms, five (4.0%) Actino-mycetes-related organisms, and 21 (16.7%) fungi. Susceptibilities to amikacin, ceftazidime, chloramphenicol, cefazolin, ciprofloxacin, gentamicin, and vancomycin are reported. CONCLUSIONS: This is the largest, single-center, prospective series on microbial susceptibilities in postoperative endophthalmitis. We report a high prevalence of gram-negative species and fungi, suggesting that empiric therapy should include coverage for gram-negative pathogens and for fungal pathogens in appropriate settings.

Microbiologic spectrum and susceptibility of isolates: part II. Posttraumatic endophthalmitis. Endophthalmitis Research Group.

PURPOSE: To present the microbial spectrum and susceptibilities of isolates in posttraumatic endophthalmitis. METHOD: Isolates from 182 eyes of 182 patients who underwent vitrectomy for posttraumatic endophthalmitis were examined. RESULTS: One hundred thirteen (62.1%) of 182 vitreous samples were culture-positive, and 23 (20.4%) of 113 culture-positive cases were polymicrobial, including three (2.7%) trimicrobial cases, yielding a total of 139 isolates. Isolates included 63 (45.3%) gram-positive cocci, 24 (17.3%) gram-positive bacilli, 25 (18.0%) gram-negative organisms, seven (5.0%) Actinomycetes-related organisms, and 20 (14.4%) fungi. Susceptibilities to amikacin, ceftazidime, chloramphenicol, cefazolin, ciprofloxacin, gentamicin, and vancomycin are reported. CONCLUSIONS: This study represents a large series on microbial spectrum and susceptibilities in posttraumatic endophthalmitis. We report a high prevalence of gram-positive bacilli species and polymicrobial infections containing gram-negative species, underscoring the importance of broad-spectrum, combination antibiotics in the empiric treatment of posttraumatic endophthalmitis.

In vitro susceptibility of bacterial keratitis pathogens to ciprofloxacin. Emerging resistance.

OBJECTIVE: To examine in vitro susceptibility of bacterial keratitis pathogens to ciprofloxacin. DESIGN: Retrospective review. PARTICIPANTS: The authors examined in vitro susceptibility of 1558 corneal isolates from 1303 patients with culture-proven bacterial keratitis seen at the LV Prasad Eye Institute in Hyderabad, India, during the 6-year period between March 1, 1991, and June 30, 1997. RESULTS: Of 1558 corneal isolates, 478 (30.7%) were not sensitive to ciprofloxacin. Among the isolates, 355 (32.5%) of the 1091 gram-positive cocci were not sensitive to ciprofloxacin, and 2 (10%) of the 20 gram-positive bacilli, 22 (13.3%) of the 165 gram-negative organisms, and 99 (35.1%) of the 282 Actinomycetes and related organisms were not sensitive to ciprofloxacin. Results from chi-square for trends analysis showed a trend of significantly increasing ciprofloxacin insensitivity in bacteria between 1992 and 1997 (P = 0.011). CONCLUSION: This is the first report of significantly increasing ciprofloxacin insensitivity among corneal pathogens. Although the lowered cost and convenience of dispensing a single, commercially available antibiotic such as ciprofloxacin in the initial treatment of bacterial keratitis is desirable, the emergence of ciprofloxacin resistance is a significant finding in this series, and the clinician should proceed with caution in the initial empiric treatment of bacterial keratitis with ciprofloxacin.

Microbial keratitis in children.

OBJECTIVE: Microbial keratitis is a major cause of corneal blindness worldwide. This problem is particularly relevant to children, because most of their visual life is ahead of them, and they are uniquely at risk for irreversible ocular deficits, such as those resulting from amblyopia. The objective of this study was to determine the etiologic agents and predisposing factors in childhood infectious keratitis and to examine the outcome of treatment in terms of structure and visual acuity. DESIGN: The study design was a retrospective cases series. PARTICIPANTS: The authors studied 113 eyes in 107 children 16 years of age and younger who were treated for (nonviral) microbial keratitis at the LV Prasad Eye Institute in Hyderabad, India, during the 4.5-year period between February 1, 1991, and June 30, 1995. INTERVENTION: The patients who met the following criteria were included in the study: (1) corneal stromal infiltrate was present on slit-lamp examination; and (2) a corneal scraping was taken at the time of examination for suspected microbial keratitis. MAIN OUTCOME MEASURES: Etiologic micro-organisms, predisposing factors, treatment method, structural treatment outcome, and visual acuity treatment outcome of the infectious keratitis episode were measured. RESULTS: The principal predisposing factors identified in this study were trauma (21.2%), ocular disease (17.7%), systemic disease (15.9%), and prior penetrating keratoplasty in the same eye (8.8%). Vitamin A deficiency was an important factor within the category of severe systemic disease, and contact lens wear was not involved in any of the cases. A total of 85 organisms were isolated in cultures of corneal scrapings from 64 (56.6%) of the 113 cases. Staphylococcus species (43.7%), Streptococcus pneumoniae (18.8%), and fungi (17.2%) were the most common isolates. Eighteen eyes (15.9%) required surgery, and 28 (36.4%) of the 77 patients on whom visual acuity was assessed at last follow-up achieved an unaided visual acuity of 20/60 or better at last follow-up. CONCLUSION: This work represents the largest recent study on childhood (nonviral) microbial keratitis, its management, and treatment outcomes. In this study, amblyopia is highlighted as a potentially significant sequela of childhood microbial keratitis. Identification of the appropriate predisposing factors, etiologic microbial organisms, and treatment outcome from this study may aid in early recognition and treatment of microbial keratitis in children.

Immunization of BALB/c mice with mIFN-gamma-secreting Mycobacterium bovis BCG provides early protection against Leishmania major infection.

We developed and tested IFN-gamma-expressing Mycobacterium bovis, strain BCG, for the ability to activate macrophages and protect mice against a heterologous challenge with Leishmania major. One, 2 or 3 weeks after intraperitoneal immunization, mice were challenged with 10(6) L. major amastigotes injected into the right footpad. Recombinant BCG immunization for all 3 challenge time points initially showed greater protection compared to the BCG control, as judged by footpad thickness and number of parasites in the leishmanial lesion. However, at week 4 after challenge, while the 1- and 2-week immunization groups continued to show increased protection, the 3-week immunization group animals exhibited progressive disease. These data suggest that the IFN-gamma-expressing BCG initially activates macrophages more effectively than native BCG, but that late exacerbation of disease can occur, highlighting the complexity of the immune response against leishmaniasis.

Secretion of human interleukin 2 by recombinant Mycobacterium bovis BCG.

The human interleukin 2 (huIL-2) gene was introduced into Mycobacterium bovis BCG by using the integrative vector pMV306. To express and secrete huIL-2 from BCG, two different plasmids, CI and CII, were made. In CI, the huIL-2-encoding region was under the control of the alpha-antigen promoter of BCG; in CII, the expression of huIL-2 was regulated by the heat shock protein 60 promoter. A signal peptide sequence isolated from the naturally secreted alpha-antigen of BCG was inserted between the promoter and huIL-2-encoding region to facilitate secretion. Both huIL-2 expression plasmids were integrated into the BCG genome when introduced into the BCG Pasteur strain by electroporation. Approximately 150 U of huIL-2 was secreted into the medium of a BCG-CII culture, while the BCG-CI cells secreted approximately one-sixth of that amount. When the IL-2-expressing BCG strain BCG-CII was injected intravenously into BALB/c mice, the number of BCG cells in the spleens of these mice was significantly less than the number in the control mice. The decreased number of IL-2-expressing BCG cells is likely due to the augmentation of the host immune response by the secreted huIL-2, although the exact mechanism is not known.

Purification, crystallization and preliminary X-ray analysis of murine interleukin-5.

Wild-type and mutant forms of murine interleukin-5 (mIL-5) have been expressed in the baculovirus expression system, purified, and used in crystallization trials. Attempts to obtain diffraction quality crystals of wild-type protein were unsuccessful. The substitution of glutamine for Asn75 preserved biological activity, while removing one of two predicted N-linked glycosylation sites, and the resulting protein was crystallized from polyethylene glycol 8000 at pH 7.8 in two crystal forms. The orthorhombic crystals, which belong to space group P2(1)2(1)2 with cell dimensions a = 55.9 A, b = 83.0 A and c = 52.3 A, diffract to beyond 2.5 A resolution. The second crystal form belongs to a trigonal space group, either P3(1)21 or P3(2)21, with cell dimensions a = b = 62.1 A, c = 129.9 A, and diffracts to about 3.8 A resolution. Each crystal form probably contains one mIL-5 dimer per asymmetric unit.

Molecular analysis of double isotype expression in IgA switching.

In this and previous studies we have shown that during the course of B cell isotype differentiation occurring in the CH12.LX B cell line, small numbers of cells appear that bear both membrane (m)IgM and membrane IgA. We have cloned relatively stable populations of such dual-bearing cells and have performed studies to determine their molecular status. In the study of one such subclone (CH12.LX.7.10), we show by primer extension that both mu and alpha mRNA contain identical VHDJH gene segments. In complementary studies of another such subclone (CH12.LX.7.13), we show using DNA restriction fragment analysis, that the C mu gene segment, but not the C alpha gene segment, is associated with the JH gene segment. Additionally, Northern blot analysis of productive message of this clone shows that both mu and alpha mRNA cohybridize to the same VH region-specific probe. Taken together, these data suggest that CH12.LX mIgM/mIgA dual-bearing cells occur in the absence of C mu gene segment deletion and that the mu and alpha mRNA transcripts are derived using an alternative mechanism such as transplicing or alternative splicing of a long RNA transcript. On the assumption that these clones are representative of mIgM/mIgA dual-bearing cells appearing during CH12.LX IgM to IgA isotype differentiation, these data support the idea that B cells simultaneously produce mu and alpha mRNA transcripts during one stage of IgA isotype switching.

The roles of IL-4, TGF-beta and LPS in IgA switching.

CH12.LX B cells have been used as a lymphoma model of MHC restricted, antigen-dependent B cell differentiation. These B cells express surface IgM and secrete IgM. Most recently we have demonstrated that CH12.LX is a model of cytokine driven IgA differentiation. Recently, transforming growth factor beta (TGF-beta) has been shown to be a probable switch factor for IgA in LPS-stimulated mouse lymphocytes, therefore we chose CH12.LX B cells to study the effect of IL-4, TGF-beta and LPS in IgA isotype switching. Adding TGF-beta to the monoclonal cell line CH12.LX results in induction of mIgA expression but no enhancement of IgA secretion similar to the effect of IL-4. The addition of LPS serves as a non-specific stimulus to enhance the secretion of the expressed immunoglobulin, but has no IgA specific activity of its own. IL-4 and TGF-beta together are synergistic for mIgA expression. Pretreatment studies show that TGF-beta added after IL-4 is the same as TGF-beta alone whereas the converse is the same as adding both cytokines together. TGF-beta acts by increasing the steady state levels of alpha message, whereas northern analysis indicates that IL-4 does not induce alpha message the way TGF-beta does. These data confirm that TGF-beta by itself is an isotype switch factor for IgA. In addition, IL-4 and TGF-beta cause mIgA expression through different mechanisms. CH12.LX B cells serve as a valuable model to study the role of multiple signals required for mIgA expression and IgA secretion.

Use of restriction fragment length polymorphisms resolved by pulsed-field gel electrophoresis for subspecies identification of mycobacteria in the Mycobacterium avium complex and for isolation of DNA probes.

Mycobacterial strains from the Mycobacterium avium complex were compared with each other and with Mycobacterium phlei isolates by restriction endonuclease digestion of chromosomal DNA with SspI and analysis by pulsed-field gel electrophoresis. Characteristic profiles were observed for known typed strains, and five groups were identified. Primary bovine isolates identified as Mycobacterium paratuberculosis by classical methods were shown to fall into both the M. paratuberculosis- and M. avium-like groups. M. paratuberculosis 18 was in the latter category. Two Mycobacterium intracellulare strains of different Schaefer serotypes had different digestion profiles. In addition, this system was exploited for the preparation of DNA probes by the isolation, digestion, and subcloning of DNA fragments separated by pulsed-field gel electrophoresis. Probe JC12 hybridized only to M. avium complex strains, but not to M. phlei, showing characteristic hybridization profiles for each of the groups previously identified by pulsed-field gel electrophoresis. The approach taken in the study lends itself to the comparative analysis of members of the M. avium complex and to the isolation and characterization of DNA probes with specificity for these mycobacteria.

High-level production of murine interleukin-5 (IL-5) utilizing recombinant baculovirus expression. Purification of the rIL-5 and its use in assessing the biologic role of IL-5 glycosylation.

We describe here a recombinant baculovirus expression system useful for high level production of murine recombinant interleukin-5 (rIL-5). In addition, we describe a single-step technique of purification of the rIL-5 from the baculovirus-infected Sf9 cell supernatants, using an anti-IL-5 affinity column. The baculovirus-derived rIL-5 has physical properties and functional activities in various lymphoid cell assays similar to those of natural T cell-derived IL-5 and reacts with anti-IL-5 antibodies. Finally, the rIL-5 is similar to natural T cell-derived IL-5 in manifesting heterogeneous glycosylation; however, glycosylation does not appear to be necessary for biologic function, at least in a lymphoid cell proliferation assay.

IL-6 is a potent cofactor of IL-1 in IgM synthesis and of IL-5 in IgA synthesis.

In these studies we determined the capacity of IL-6 to act as a differentiation cofactor for murine Peyer's patch B cells producing different Ig classes and subclasses. In preliminary studies we determined that sufficient endogenous IL-6 was produced in LPS-induced cell systems to obscure responses to exogenous IL-6. We therefore studied IL-6 effects on Peyer's patch B cells (T cell-depleted cell populations) in the absence of LPS, relying on responses of in vivo-activated cells. rIL-1 alpha or purified IL-6 only slightly enhanced synthesis of IgM over minimal baseline levels in Peyer's patch T cell-depleted cell cultures; however, when IL-6 was added to cultures also containing rIL-1, IgM synthesis was very substantially increased. In addition, rIL-5 alone gave rise to a modest increase in IgM synthesis and its effect was not enhanced by either rIL-1 or IL-6. IgG production (mainly IgG3) followed a similar pattern. In contrast, IgA production was only modestly increased above baseline by rIL-1, rIL-5, or IL-6 alone or by rIL-1 and IL-6 in combination, but was greatly increased by rIL-5 and IL-6 in combination. The effect of IL-6 on Ig synthesis in the above studies was not due to an effect on cell proliferation. In summary, these data indicate that B cells differ in respect to the cytokines supporting maximal terminal differentiation and thus the class of Ig produced may depend on the presence of a particular combination of cytokines and lymphokines.

Regulation of IgA differentiation in CH12LX B cells by lymphokines. IL-4 induces membrane IgM-positive CH12LX cells to express membrane IgA and IL-5 induces membrane IgA-positive CH12LX cells to secrete IgA.

In these studies we utilized the Ag (SRBC)-reactive B cell line CH12LX to study isotype switching. CH12LX cells are a stable population of B cells mainly bearing membrane IgM (mIgM) (98 to 99%) with a small population of B cells bearing membrane IgA (mIgA) (1 to 2%). LPS induced a 5- to 10-fold increase in the secretion of both Ig, whereas a lymphokine-rich supernatant of D10 T cells induced a greater increase in the secretion of IgA than IgM. Analysis of the latter effect with recombinant lymphokines disclosed that rIL-4 induced an increase in the number of mIgA+ cells (6 to 15%) with minimal effect on IgA secretion, whereas IL-5 induced increased IgA secretion but had no effect on mIgA expression. The addition of both lymphokines induced increased mIgA expression and IgA secretion. No effect on mIgA expression or IgA secretion was seen with other lymphokines, including IL-1, IL-2, IL-3, IL-6, GM-CSF, and IFN-gamma. The rIL-4 effect on CH12LX cells represents true differentiation rather than selective proliferation for the following reasons: first, subclones of CH12LX cells respond to IL-4-containing T cell supernatant in the same fashion as the original cell line; second, culture of CH12LX cells with IL-4 causes the appearance of large numbers of dual-bearing mIgM/mIgA cells as well as mIgA+ cells and a dual-bearing mIgM/mIgA line was obtained by cloning CH12LX after stimulation with an IL-4-containing supernatant; third, sorted mIgA+ and mIgA- CH12LX cells had similar rates of proliferation in the presence or absence of IL-4. In further studies, it was found that IL-5 causes IgA secretion by mIgA+ but not mIgA- CH12LX cells indicating that it is acting as a post-isotype switch differentiation factor. These studies are consistent with the view that IL-4 and IL-5 act in a sequential fashion to induce IgA expression and secretion in CH12LX cells, IL-4 inducing differentiation of mIgM+ cells to mIgA+ cells and IL-5 enhancing the IgA secretion by the resulting mIgA-bearing cells.