Laryngeal swabs for diagnosing tuberculosis.

The value of smear and culture of laryngeal swabs as a method of confirming pulmonary tuberculosis was investigated in Indian children. A total of 116 children with 'suspected' tuberculosis had a Mantoux test and chest X-ray. Of these, 51 had a positive Mantoux and/or chest X-ray compatible with tuberculosis, and this group had two laryngeal swabs taken on each of 3 consecutive days. The Mantoux test was positive in 37 (73%) of the 51 'probable' cases. Chest X-ray was abnormal in 36 (71%) cases and compatible with tuberculosis in 20 (39.7%). Mycobacterium tuberculosis was cultured from laryngeal swabs in 14 (28%) children and in another three children smears were positive but culture-negative. The overall confirmation rate for tuberculosis was 33%. Laryngeal swabs are a simple method of confirming tuberculosis and may be undertaken in out-patients.

Factors associated with the duration of breastfeeding: analysis of the primary and secondary responders to a self-completed questionnaire.

A cohort of 1192 consecutive newborn infants was followed prospectively for factors possibly affecting the length of time they were breastfed. Following the procedure of a double-blind test, one-third of the cohort received Credé prophylaxis at age 2 h. The duration of breastfeeding (sole or partial) was recorded up to age 6 months and there was a 100% follow-up. Multivariate proportional hazards regression analysis (Cox) of the whole cohort showed that babies being delivered between 21.00 and 24.00 h were associated with a shorter duration of breastfeeding (rate ratio = 1.37, 99% confidence interval = 1.05-1.78). Mother's age (under 21 years), marital status (unmarried) and birthweight (inversely) were factors also independently associated with shorter breastfeeding duration. Boys were breastfed for a shorter time than girls (p < 0.05). In univariate analyses only, the first-born babies had a significantly shorter breastfeeding time, and purulent eyes in the first 24 h was a factor of borderline significance (p < 0.05). Educational level, socioeconomic status and smoking habits of the mothers were not investigated in this study. Owing to the lack of regulations in place at the time of the study (1981-82), it was possible to differentiate between the mothers who responded spontaneously to the self-completion questionnaire (primary responders, 68.5%) and those who required one or two reminders. Short breastfeeding time was the strongest predictor of being a secondary responder, followed by being very young or unmarried. Approaching the secondary responders reduced the prevalence of breastfeeding at 6 months by 5% (from 53.8% to 48.8%).

First-rib exostosis bursa.

We describe a case of exostosis bursa of the first left rib. Clinical features, investigations, and surgical management are described in detail.

Direct DNA immunization of mice with plasmid DNA encoding the tegument protein pp65 (ppUL83) of human cytomegalovirus induces high levels of circulating antibody to the encoded protein.

The 65 kDa tegument protein (pp65 or rather ppUL83) of human cytomegalovirus (HCMV) has been shown to be a major cytotoxic T-lymphocyte target during natural infection, and thus appears to be an appropriate candidate to be evaluated as a component of a HCMV polynucleotide vaccine. We have constructed expression vectors pH beta-pp65, in which pp65 expression is under the control of human beta-actin promoter, and pCMVint-pp65, in which pp65 expression is driven by the HCMV immediate-early promoter along with the intron A. These construct DNAs were utilized for the intramuscular injection into the quadriceps of BALB/c mice. Approximately 60% of the mice showed the presence of anti-pp65 antibodies following the second DNA inoculation with 100 micrograms of plasmid DNA. The anti-pp65 antibody titer was higher in the group of mice that was injected with pCMVint-pp65 plasmid DNA compared to the group that was injected with pH beta-pp65 DNA, presumably due to a higher level of pp65 expression using the former plasmid.

Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes.

Primary cytomegalovirus (CMV) infection and reactivation of persistent CMV are associated with significant morbidity and mortality in immunocompromised individuals. Although recovery from CMV disease is correlated with the development of CMV-specific cytotoxic T lymphocytes (CTL), the major viral target antigens to which the response is directed are ill-defined, though they may comprise viral structural elements. We now identify the CMV matrix protein pp65 as a significant target antigen for CD8+ class I major histocompatibility complex (MHC)-restricted CMV-specific CTL derived from the peripheral blood of four of five latently infected individuals. CMV-specific CTL recognition of pp65 on target cells occurs prior to the onset of viral gene expression and persists throughout the duration of the replicative cycle. Recognition in the absence of viral gene expression suggests that abundant viral protein enters the normal trafficking pathway upon viral penetration and is readily made available to MHC molecules for presentation at the cell surface. Thus pp65 specific CTL may represent an important effector population for early control and limitation of CMV infection and disease. The observation that CMV-specific CTL can be induced in vitro using peptide fragments derived from pp65 supports the future use and manipulation of this and similar effector populations in a clinical setting.

Expression in insect cells and immune reactivity of a 28K tegument protein of human cytomegalovirus.

The gene encoding the highly antigenic 28K (pp28) tegument phosphoprotein of human cytomegalovirus (HCMV) was expressed in insect cells utilizing a recombinant baculovirus. The mature intracellular form of the recombinant-derived pp28 had mobility on SDS-polyacrylamide gels similar to that of native pp28 from HCMV strain Towne-infected human foreskin fibroblasts (HFFs). In vitro labelling of recombinant Autographa california nuclear polyhedrosis virus-infected Spodoptera frugiperda cells or of HCMV-infected HFFs with [32P]orthophosphate followed by immunoprecipitation showed that both the insect cell-derived and HCMV strain Towne-infected fibroblast-derived pp28 were phosphorylated. The mobility of pp28 derived from these two sources as well as from extracellular HCMV virions indicated the existence of multiple charged forms of the protein, and a difference in the relative amounts of these forms expressed in HCMV-infected HFFs and recombinant baculovirus-infected insect cells. The recombinant pp28 expressed in insect cells was readily and specifically recognized by antibodies to native pp28, including HCMV-seropositive human serum, and was used in an ELISA to screen human sera for seropositivity.

Concomitant occurrence of mucopolysaccharidosis IIIB and Glanzmann's thrombasthenia. Further evidence of a hyperactive alpha-N-acetylglucosaminidase-producing allele.

A daughter of first cousins had two extremely rare, recessive disorders: thrombasthenia (Glanzmann's disease, glycoprotein IIb/IIIa deficiency) and mucopolysaccharidosis IIIB, (Sanfilippo B syndrome, alpha-N-acetylglucosaminidase (NAG) deficiency). Normal alpha-N-acetylglucosaminidase activity was observed in two obligate heterozygotes (the proband's father and her maternal grandmother), suggesting that in addition to the normal and defective alleles, a third, hyperactive allele is also present in this family. Such a hyperactive allele seems to be quite prevalent in our area, and makes the biochemical identification of heterozygotes impossible if no extensive family surveys provide additional clues. There was no linkage between the two diseases, nor between any of them and several blood-groups and HLA-antigens tested for.

Human cytomegalovirus strain Towne pp28 gene: sequence comparison to pp28 of HCMV AD169 and stable expression in Chinese hamster ovary cells.

Human cytomegalovirus (HCMV) contains a 28-kDa (pp28) matrix phosphoprotein which has been shown to be highly immunogenic in humans. We have cloned and sequenced the gene encoding pp28 of HCMV Towne strain (pp28Towne) and have expressed this gene in stable Chinese hamster ovary (CHO) cell lines in order to examine the structural, functional, and antigenic properties of this protein. The pp28Towne gene had 99% nucleotide and 98.4% amino acid similarity to the pp28 gene of HCMV AD169 strain (pp28AD169). We identified three amino acid substitutions (Gly70 to Ser70, Ser76 to Asn76, and Thr85 to Ala85) in pp28Towne, all clustered in a short 16 amino acid stretch located in the N-terminal half of the protein. The pp28Towne gene was expressed in CHO cells using a vector in which transcription was driven by a human beta-actin promoter. The expressed protein, having an electrophoretic mobility similar to that of HCMV-derived pp28, reacted strongly in immunoblot analysis with pp28-specific murine monoclonal antibodies as well as HCMV-seropositive human sera.

Characterization of a 52K protein of murine cytomegalovirus and its immunological cross-reactivity with the DNA-binding protein ICP36 of human cytomegalovirus.

We have developed a hybridoma, designated 25G11, which produced a monoclonal antibody (MAb) reactive with a 52K protein of murine cytomegalovirus (MCMV). This MAb, 25G11, was reactive with a protein band of 52K in MCMV-infected cell lysates and with a protein of 49K in human CMV (HCMV)-infected cell lysates as detected by immunoblot analysis. With purified MCMV virions, 25G11 gave a faintly immunoreactive band of 52K. However, no immunoreactive protein band was detected with purified HCMV virions, nor with purified HCMV or MCMV envelope preparations. By immunocytochemistry, 25G11 detected viral antigen primarily in the nucleus of HCMV- or MCMV-infected cells. The antibody 25G11 was used to screen a lambda gt11 library of HCMV DNA fragments. One of the isolated clones (lambda 32323B) was employed for gene mapping on the HCMV genome, which suggested that the immunoreactive HCMV protein was the DNA-binding protein (ICP36). Analysis of the recombinant fusion protein with antibody 25G11 and with an MAb (CH16) specific for an HCMV DNA-binding protein confirmed the identity of the cross-reacting protein as ICP36. Furthermore, we found that whereas the epitope recognized by 25G11 was conserved between HCMV and MCMV proteins, the epitope recognized by CH16 was unique to HCMV and thus represents a variable region in the protein.

Human cytomegalovirus strain Towne pp65 gene: nucleotide sequence and expression in Escherichia coli.

The human cytomegalovirus (HCMV) encodes a 65-kDa tegument protein (pp65), which has been reported to be a target of immune response during natural infection. We have cloned and sequenced the gene encoding pp65 of HCMV Towne strain (pp65Towne), and have expressed this gene in Escherichia coli in order to study certain antigenic and structural properties of this polypeptide. The pp65Towne gene had a 99% nucleotide similarity and 99.7% amino acid similarity to pp65 of HCMV AD169 strain (pp65AD169). However, unlike the pp65AD169 gene, the pp65Towne gene was found to be incapable of undergoing RNA splicing due to a base substitution in the critical 3' splice-acceptor site. Insertion of this protein coding sequence into the bacterial expression plasmids enabled synthesis in E. coli of an immunoreactive pp65-related polypeptide. The recombinant pp65 (rpp65) reacted strongly in immunoblot analysis with pp65-specific murine and human monoclonal antibodies as well as with anti-pp65 rabbit antiserum. In immunoblot analysis, the reactivity of rpp65 with a panel of human HCMV-immune sera indicated that some sera were reactive while other HCMV seropositive sera were nonreactive, a finding similar to that for native pp65.

Human plasma fibronectin. Demonstration of structural differences between the A- and B-chains in the III CS region.

Fibronectins are a class of cell-adhesion proteins produced from a single gene. The soluble plasma form is synthesized by hepatocytes and the insoluble cellular form by fibroblasts and other cell types. The proteins possess multiple binding domains for macromolecules including collagen, fibrin and heparin along with at least one cell-binding domain. Cellular as well as plasma fibronectins are dimers of similar but not identical polypeptides. Their differences are the result of internal amino acid sequence variability due to alternative RNA splicing in at least three regions (ED-A, ED-B and III CS). We have been studying this polymorphism at the protein level in plasma fibronectin (pFn). Cathepsin D-digested pFn applied to a heparin-agarose column and eluted with an NaCl stepwise gradient (0.1 M, 0.25 M and 0.5 M) released two polypeptides (75 kDa and 65 kDa) in the 0.5 M-NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the C-terminal heparin-binding domain) and AHB-3 (specific for the III CS domain) suggest that both peptides contain the C-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the III CS region. These two polypeptides (75 kDa and 65 kDa) were digested with trypsin, and the resulting peptides were analyzed by fast-atom-bombardment mass spectrometry and compared with the known cDNA-derived peptide sequence. Peptides that were unique to the III CS region were further characterized by micro sequence analysis. The 75 kDa fragment is derived from the A-chain and contains the III CS region (89 amino acid residues) along with the C-terminal heparin-binding (Hep-2) domain and the fibrin-binding (Fib-2) domain. A single galactosamine-based carbohydrate group was detected at Thr-73/74 of the III CS region present in the 75 kDa fragment. The 65 kDa fragment is derived from the B-chain and lacks the entire III CS region but does contain the Hep-2 and Fib-2 domains.

Comparative analysis of human cytomegalovirus a-sequence in multiple clinical isolates by using polymerase chain reaction and restriction fragment length polymorphism assays.

The human cytomegalovirus (HCMV) a-sequence (a-seq) is located in the joining region between the long (L) and short (S) unique sequences of the virus (L-S junction), and this hypervariable junction has been used to differentiate HCMV strains. The purpose of this study was to investigate whether there are differences among strains of human cytomegalovirus which could be characterized by polymerase chain reaction (PCR) amplification of the a-seq of HCMV DNA and to compare a PCR method of strain differentiation with conventional restriction fragment length polymorphism (RFLP) methodology by using HCMV junction probes. Laboratory strains of HCMV and viral isolates from individuals with HCMV infection were characterized by using both RFLPs and PCR. The PCR assay amplified regions in the major immediate-early gene (IE-1), the 64/65-kDa matrix phosphoprotein (pp65), and the a-seq of the L-S junction region. HCMV laboratory strains Towne, AD169, and Davis were distinguishable, in terms of size of the amplified product, when analyzed by PCR with primers specific for the a-seq but were indistinguishable by using PCR targeted to IE-1 and pp65 sequences. When this technique was applied to a characterization of isolates from individuals with HCMV infection, selected isolates could be readily distinguished. In addition, when the a-seq PCR product was analyzed with restriction enzyme digestion for the presence of specific sequences, these DNA differences were confirmed. PCR analysis across the variable a-seq of HCMV demonstrated differences among strains which were confirmed by RFLP in 38 of 40 isolates analyzed. The most informative restriction enzyme sites in the a-seq for distinguishing HCMV isolates were those of MnlI and BssHII. This indicates that the a-seq of HCMV is heterogeneous among wild strains, and PCR of the a-seq of HCMV is a practical way to characterize differences in strains of HCMV.

Structural analysis of a 64-kDa major structural protein of human cytomegalovirus (Towne): identification of a phosphorylation site and comparison to pp65 of HCMV (AD169).

The major 64-kDa structural protein of human cytomegalovirus (pp64) was isolated from a guanidinium chloride extract of the virions and dense bodies of HCMV (Towne) by reverse-phase HPLC. Purified pp64 was reduced and alkylated followed by digestion with trypsin. The molecular mass of each of the tryptic peptides was determined by fast atom bombardment/mass spectrometry and compared with the predicted molecular mass of the fragments deduced from the corresponding DNA-derived peptide sequence of pp65 from HCMV (AD169). Microsequence analysis was employed to confirm selected peptides. Results of protein sequence analysis of pp64 from HCMV (Towne) are in complete agreement with the DNA-derived protein sequence of pp65 predicted for HCMV (AD169) with the following exceptions and modifications. The protein isolated from HCMV (Towne) was found to contain an Ala at position 448 instead of Ser448 reported for the protein from HCMV (AD169). We also identified Ser472 as a site of phosphorylation in pp64 from HCMV (Towne). Finally, on the basis of the sequence of HCMV (AD169) DNA fragment encoding the matrix protein and on S1 nuclease protection analysis, it has been predicted that one version of the matrix protein (possibly the lower matrix protein, Mr 65K) is encoded by an mRNA that is formed through splicing of a short intron. However, we have obtained peptides that contain sequences spanning through the splice-junction region, suggesting that in HCMV (Towne), the matrix protein is encoded by an unspliced message.

Altered expression of fibronectin gene in cells infected with human cytomegalovirus.

Human cytomegalovirus (HCMV) induces morphological changes in infected cells that are remarkably similar to those seen in oncogenically transformed cells. The molecular bases of these phenotypic alterations are not known but their occurrence in some transformed cells can be associated with abnormal fibronectin (FN) expression. In this report, we have compared FN levels in normal and HCMV-infected cells. In these studies, the HCMV-infected fibroblasts exhibited a progressive loss of cellular FN. Northern (RNA) blot analysis revealed that the decrease in FN levels resulted from a lowering of FN mRNA levels in HCMV-infected cells. We detected an initial decrease in FN mRNA of 25 to 30% at immediate-early and early times, whereas at late times after infection the levels of FN mRNA were lowered by greater than 80%. These results indicated that the HCMV-induced decrease in FN expression is due to a decrease in the quantity of FN mRNA and suggested that HCMV-encoded and/or -induced functions may be involved in producing these alterations.

Genomic locus for a 140 kDa structural protein (pp150) of human cytomegalovirus in strains Towne and AD169.

An HCMV specific clone was isolated from a genomic library of human cytomegalovirus (HCMV) DNA cloned into the expression vector lambda gt11. This clone (lambda 111-1) expressed an HCMV/beta-galactosidase fusion protein which was reactive with rabbit antibody prepared against purified HCMV virions and dense bodies as well as human HCMV immune serum. By probing Western blots of HCMV virion proteins or HCMV-infected cells with antibody prepared against the fusion protein, the authentic gene product of clone lambda 111-1 was identified as a high molecular weight polypeptide of 140. Probing the restriction digests of HCMV DNA with insert DNA from the immunoreactive lambda gt11 clone permitted us to localize the coding sequence for the 140 kDa polypeptide to the long unique region (map coordinates of 0.16-0.18) on HCMV Towne and AD169 genomes.

Genomic localization of the gene encoding a 32-kDa capsid protein of human cytomegalovirus.

We have determined the map position of a viral gene encoding a 32-kDa late structural protein of human cytomegalovirus (HCMV) using a murine monoclonal antibody. This monoclonal antibody was reactive with two protein bands of 32 and 27 kDa in HCMV-infected cell lysates and with a single 32-kDa protein band in HCMV virions as detected by immunoblot analysis. When purified HCMV envelope preparation was used for immunoblotting, the monoclonal antibody did not display a detectable band. We used this monoclonal antibody to screen a cDNA library that was constructed from poly(A)+ RNA of late HCMV-infected cells and cloned into the expression vector lambda gt11. A cDNA clone that expressed an immunoreactive epitope of the late HCMV protein fused to beta-galactosidase was identified. Probing the restriction digests of HCMV (Towne and AD169) DNA with insert DNA from the immunoreactive lambda gt11 clone permitted us to localize the coding sequence within the long unique region between map coordinates of 0.62 and 0.64 of HCMV Towne and AD169 genomes. Using the same probe, a single transcript of 1.4 kb was detected in total RNA from HCMV-infected cells at late times after infection.