Resistance of normal serum IgA and secretory IgA to bacterial IgA proteases: evidence for the presence of enzyme-neutralizing antibodies in both serum and secretory IgA, and also in serum IgG

Normal serum IgA and secretory IgA (sIgA) of subclass IgA1 were isolated from pooled human serum and milk, respectively. They were tested for their susceptibility to bacterial IgA proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria gonorrhoeae, and Neisseria meningitidis that cleave IgA of only the IgA1 subclass. They were also tested for susceptibility to a novel IgA-protease from Clostridium ramosum that cleaves IgA of the IgA1 as well as the IgA2 subclass of the A2m(1) allotype. Both normal serum IgA1 and sIgA1 exhibited resistance to most IgA proteases. The one exception was the IgA protease from C. ramosum which readily cleaved both the serum IgA1 and sIgA1 into Fab and Fc fragments. Secretory component (SC) had nothing to do with the resistance of these IgAs. The resistance of these IgAs to most of the IgA proteases was found to be due to their enzyme-neutralizing antibody activity, since the Fab but not the Fc fragment of sIgA1 showed enzyme-inhibitory activity against these IgA proteases. Similar enzyme-neutralizing antibody activity was found in the pepsin-digested normal serum IgG-(Fab')2 fragment. These results indicate that the induction of the enzyme-neutralizing antibodies against the bacterial IgA proteases took place not only in mucosal sIgA but also in serum IgA and IgG. No enzyme-neutralizing antibody activity against the novel IgA-protease of C. ramosum was detected in any immunoglobulin preparations used in the present study or in the serum of a patient who carries the IgA protease-producing strain of C. ramosum in his feces.     

Sites in the CH3 domain of human IgA1 that influence sensitivity to bacterial IgA1 proteases

The influence of regions, other than the hinge, on the susceptibility of human IgA1 to cleavage by diverse bacterial IgA1 proteases, was examined using IgA1 mutants bearing amino acid deletions, substitutions, and domain swaps. IgA1 lacking the tailpiece retained its susceptibility to cleavage by all of the IgA1 proteases. The domain swap molecule alpha1alpha2gamma3, in which the CH3 domain of IgA1 was exchanged for that of human IgG1, was resistant to cleavage with the type 1 and 2 serine IgA1 proteases of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae, but remained sensitive to cleavage with the metallo-IgA1 proteases of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis. Substitution of the IgA1 Calpha3 domain motif Pro440 -Phe443 into the corresponding position in the Cgamma3 domain of alpha1alpha2gamma3 resulted now in sensitivity to the type 2 IgA1 protease of N. meningitidis, indicating the possible requirement of these amino acids for sensitivity to this protease. For the H. influenzae type 2 protease, resistance of an IgA1 mutant in which the CH3 domain residues 399-409 were exchanged with those from IgG1, but sensitivity of mutant HuBovalpha3 in which the Calpha3 domain of bovine IgA replaces that of human IgA1, suggests that CH3 domain residues Glu403, Gln406, and Thr409 influence sensitivity to this enzyme. Hence, unlike the situation with the metallo-IgA1 proteases of Streptococcus spp., the sensitivity of human IgA1 to cleavage with the serine IgA1 proteases of Neisseria and Haemophilus involves their binding to different sites specifically in the CH3 domain.     

The influences of hinge length and composition on the susceptibility of human IgA to cleavage by diverse bacterial IgA1 proteases

The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val(222) and Cys(241) (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc.     

Effect of IgA1 Protease on the Ability of Secretory IgA1 Antibodies to Inhibit the Adherence of Streptococcus mutans

Secretory IgA (SIgA) is the principal immunoglobulin isotype present in the mucosal secretions of humans. SIgA is thought to play a major role in host defense at these surfaces by inhibiting the colonization of potentially pathogenic microorganisms. A number of bacteria that are mucosal pathogens of humans produce a protease that specifically cleaves the IgA1 subclass of humans and great apes at the hinge region to produce Fab and Fc fragments. In order to study the effect of IgA1 protease on the ability of SIgA1 antibodies to inhibit bacterial adherence, an in vitro assay that quantifies the adsorption of radiolabeled Streptococcus mutans to hydroxyapatite (HA) beads was employed. High titer S. mutans-specific SIgA1 and SIgA2 antibodies were induced in chimpanzee milk for use in the assay. Fabα1 fragments had significantly reduced ability to inhibit adherence of S. mutans to saliva-coated HA compared to intact SIgA1 or SIgA2 anti-S. mutans antibodies. These data support the potential importance of IgA1 proteases as an ecological determinant in the oral cavity and their role as a determinant of pathogenesis of pathogenic bacteria whose portal of entry is the mucosal surface.     

IgA1 proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Streptococcus sanguis: comparative immunochemical studies

IgA1 proteases from H. influenzae, N. meningitidis, S. pneumoniae, and S. sanguis were compared with respect to site of cleavage in the IgA1 molecule and EDTA sensitivity. Proteases from S. sanguis and S. pneumoniae cleaved the Pro (227)-Thr (228) bond within the hinge region of the alpha 1 chain and were inhibited by EDTA. H. influenzae IgA1 protease cleaved the Pro (231)-Ser (232) peptide bond. The activity of IgA1 proteases from H. influenzae and N. meningitidis was unaffected by EDTA. Purified and denatured alpha 1 chain was cleaved only in the hinge region. Other component chains of secretory IgA (secretory component, light and J chains) were not susceptible. In addition to IgA1 protease, S. pneumoniae released exo- and endoglycosidases that removed a considerable portion of carbohydrate side chains of IgA1; this activity was absent from crude IgA1 protease preparations of the other three bacterial species. Association in vitro of polymeric IgA1 with SC did not inhibit the degradation of IgA1 proteases. The considerable resistance of secretory IgA to cleavage by IgA1 proteases may be explained in part by the presence of IgA1 protease-neutralizing antibodies in secretory IgA.     

Disulfide bonding of secretory component to a single monomer subunit in human secretory IgA.

The arrangement of disulfide bonds joining secretory component (SC) to the alpha chains in secretory IgA was studied by determining the molecular size of the principal fragments resulting from CNBr digestion of secretory dimeric Fc fragments from IgA (Fc)2alpha fragments). In vitro complexes formed by incubating 125I-free SC and myeloma 131I-(Fc)2alpha fragments were isolated by gel filtration and subsequently digested with cyanogen bromide. The CNBr digests of SC-(Fc)2alpha fragments were analyzed by gel filtration in 5 M guanidine. Two principal fragments were obtained, one containing a monomeric Fc fragment from IgA (Fcalpha) associated with SC (m.w. congruent to 110,000) and a second containing the second Fcalpha monomer (m.w. congruent to 50,000) from the dimeric SC-(Fc)2alpha. Similar results were obtained when secretory (Fc)2alpha fragments isolated from native secretory IgA dimer were subjected to CNBr digestion. The data indicate that SC is disulfide bonded to a single monomer subunit in secretory IgA dimer.     

Bacterial immunoglobulin A proteases monitored by continuous spectrophotometry

IgA proteases were estimated in a turbid aqueous two-phase system with 10% polyethylene glycol-Tris buffer, where IgA spontaneously concentrates in microscopic spherical particles (less than 1 micron). After enzymatic cleavage of IgA into Fab alpha and Fc alpha fragments, these fragments are soluble and decreasing turbidity is observed. The reaction may be followed by conventional spectrophotometry. In this manner, IgA proteases may be estimated in 10 min. Examples of the utility of the method are given with results from inhibitor studies, estimation of Km and purification of IgA protease from Haemophilus influenzae.     

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.     

Clostridium ramosum, an IgA protease-producing species and its ecology in the human intestinal tract

A bacterial strain isolated from feces of a patient with ulcerative colitis, which had been shown to produce a novel immunoglobulin A (IgA) protease (cleaving both the human IgA1 subclass and IgA2 subclass of A2m(1) allotype) extracellularly, was identified as Clostridium ramosum. By using a selective medium (propionate-rifampicin-gentamicin-colimycin-polymyxin medium) devised for C. ramosum, analysis of the population level of this organism was performed to determine its ecology in the human intestinal tract. C. ramosum was isolated in 20 of 25 fecal samples (80%) from patients with inflammatory bowel disease (I.B.D.) and in 112 of 135 samples (83%) from patients without I.B.D. (control group). C. ramosum was also isolated from 6 of 11 biopsy samples (55%) of the inflamed rectal mucosa from patients with ulcerative colitis and from five of 15 samples (33%) from the intact mucosa of the control group. The population levels of C. ramosum in most of the biopsy samples ranged from 2.3 to 5.0 log10 per gram. The IgA protease-positive C. ramosum was found in only four of 135 fecal samples (3%) and one of 15 biopsy samples (6.7%) from the control group. These results indicate that IgA protease-positive C. ramosum is not likely to play a role in the induction of I.B.D., unless the organism is first isolated from the patient with I.B.D.     

Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1

Immunoglobulin A (IgA), the most abundant human immunoglobulin, mediates immune protection at mucosal surfaces as well as in plasma. It exists as two subclasses IgA1 and IgA2, and IgA2 is found in at least two allotypic forms, IgA2m(1) or IgA2m(2). Compared to IgA1, IgA2 has a much shorter hinge region, which joins the two Fab and one Fc fragments. In order to assess its solution structure, monomeric recombinant IgA2m(1) was studied by X-ray and neutron scattering. Its Guinier X-ray radius of gyration R(G) is 5.18 nm and its neutron R(G) is 5.03 nm, both of which are significantly smaller than those for monomeric IgA1 at 6.1-6.2 nm. The distance distribution function P(r)for IgA2m(1) showed a broad peak with a subpeak and gave a maximum dimension of 17 nm, in contrast to the P(r) curve for IgA1, which showed two distinct peaks and a maximum dimension of 21 nm. The sedimentation coefficients of IgA1 and IgA2m(1) were 6.2S and 6.4S, respectively. These data show that the solution structure of IgA2m(1) is significantly more compact than IgA1. The complete monomeric IgA2m(1) structure was modelled using molecular dynamics to generate random IgA2 hinge structures, to which homology models for the Fab and Fc fragments were connected to generate 10,000 full models. A total of 104 compact best-fit IgA2m(1) models gave good curve fits. These best-fit models were modified by linking the two Fab light chains with a disulphide bridge that is found in IgA2m(1), and subjecting these to energy refinement to optimise this linkage. The averaged solution structure of the arrangement of the Fab and Fc fragments in IgA2m(1) was found to be predominantly T-shaped and flexible, but also included Y-shaped structures. The IgA2 models show full steric access to the two FcalphaRI-binding sites at the Calpha2-Calpha3 interdomain region in the Fc fragment. Since previous scattering modelling had shown that IgA1 also possessed a flexible T-shaped solution structure, such a T-shape may be common to both IgA1 and IgA2. The final models suggest that the combination of the more compact IgA2m(1) and the more extended IgA1 structures will enable human IgA to access a broader range of antigens than either acting alone. The hinges of both IgA subclasses appear to show reduced flexibility when compared to their equivalents in IgG, and this may be important for maintaining an extended IgA structure.     

IgA protease activity of microbes in the genus Bordetella

Monoclonal IgA paraproteins of subclasses 1 and 2, isolated from the sera of myeloma patients, were incubated for 4, 24, 48 and 72 hours with B. pertussis, B. parapertussis, B. bronchiseptica cultures, as well as Haemophilus influenzae strain. The fragmentation of IgA was studied by immunielectrophoresis with antisera to alpha-chain, to Fab alpha + Fc alpha, to Fab alpha and with antisera to light chains corresponding to the type of paraprotein. B. pertussis and B. parapertussis were found to have subclass-unspecific IgA protease which splitted off a cathode fragment, similar to Fab-fragment and, probably, corresponding to the variable domain of alpha-chain (Fv), after 48-hour incubation. Similar IgA protease was detected in H. influenzae, found to have classical IgA1 protease as well. All Bordetella species under study splitted off anode components from IgA paraproteins of both subclasses. These components, containing the determinants of heavy and light IgA chains, were either IgA - alpha I-antitrypsin complexes or some IgA fragments with high electrophoretic motility. None of the strains under study splitted monoclonal IgG.     

Characterization of a mutated IgA2 antibody of the m(1) allotype against the epidermal growth factor receptor for the recruitment of monocytes and macrophages

IgA antibodies constitute an important part of the mucosal immune system, but their immunotherapeutic potential remains rather unexplored, in part due to biotechnological issues. For example, the IgA2m(1) allotype carries an unusual heavy and light chain pairing, which may confer production and stability concerns. Here, we report the generation and the biochemical and functional characterization of a P221R-mutated IgA2m(1) antibody against the epidermal growth factor receptor (EGFR). Compared with wild type, the mutated antibody demonstrated heavy chains covalently linked to light chains in monomeric as well as in joining (J)-chain containing dimeric IgA. Functional studies with wild type and mutated IgA2m(1) revealed similar binding to EGFR and direct effector functions such as EGFR down-modulation and growth inhibition. Furthermore, both IgA molecules triggered similar levels of indirect tumor cell killing such as antibody-dependent cell-mediated cytotoxicity (ADCC) by isolated monocytes, activated polymorphonuclear cells, and human whole blood. Interestingly, the dimeric IgA antibodies demonstrated higher efficiency in direct as well as in indirect effector mechanisms compared with their respective monomeric forms. Both wild type and mutated antibody triggered effective FcαRI-mediated tumor cell killing by macrophages already at low effector to target cell ratios. Interestingly, also polarized macrophages mediated significant IgA2-mediated ADCC. M2 macrophages, which have been described as promoting tumor growth and progression, may convert to ADCC-mediating effector cells in the presence of EGFR-directed antibodies. In conclusion, these results provide further insight into the immunotherapeutic potential of recombinant IgA antibodies for tumor immunotherapy and suggest macrophages as an additional effector cell population.     

Primary structure of a human IgA1 immunoglobulin. IV. Streptococcal IgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain.

In order to establish the complete amino acid sequence of the human IgA alpha1 chain Bur, IgA1 protease from Streptococcus sanguis was employed to generate Fabalpha and Fcalpha fragments in the final stage of this investigation. Cyanogen bromide cleavage of the Fabalpha fragment followed by reduction and aminoethylation produced the Fd' fragment (residues 84 to 227); this contains part of the variable region (VR), the whole first constant domain (Calpha1), and part of the hinge region of this heavy chain. The tryptic peptides of the Fd' fragment were isolated, characterized, and sequenced. The results together with the data in the preceding papers on chymotryptic, tryptic, and thermolysin peptides permitted the complete amino acid sequence of the human IgA alpha1 chain to be established.     

Immunoglobulin A (IgA) is a natural ligand of hepatitis A virus cellular receptor 1 (HAVCR1), and the association of IgA with HAVCR1 enhances virus-receptor interactions

The hepatitis A virus cellular receptor 1 (HAVCR1/TIM1), a member of the T-cell immunoglobulin mucin (TIM) family, is an important atopy susceptibility gene in humans. The exact natural function of HAVCR1/TIM1 and the inverse association between HAV infection and prevention of atopy are not well understood. To identify natural ligands of human HAVCR1/TIM1, we used an expression cloning strategy based on the binding of dog cells transfected with a human lymph node cDNA library to a HAVCR1/TIM1 Fc fusion protein. The transfected cells that bound to the human HAVCR1/TIM1 Fc contained cDNA of human immunoglobulin alpha 1 heavy (Igalpha1) and lambda light (Iglambda) chain and secreted human IgA1lambda antibody that bound to the cell surface. Cotransfection of the isolated Igalpha1 and Iglambda cDNAs to na?ve dog cells resulted in the secretion of IgA1lambda that bound to HAVCR1/TIM1 Fc but not to a poliovirus receptor Fc fusion protein in a capture enzyme-linked immunosorbent assay. The interaction of HAVCR1/TIM1 with IgA was inhibited by monoclonal antibodies (MAbs) against Igalpha1 and Iglambda, excess IgA1lambda, or anti-HAVCR1/TIM1 MAb. IgA did not inhibit HAV infection of African green monkey cells, suggesting that the IgA and the virus binding sites are in different epitopes on HAVCR1/TIM1. IgA enhanced significantly the neutralization of HAV by HAVCR1/TIM1 Fc. Our results indicate that IgA1lambda is a specific ligand of HAVCR1/TIM1 and that their association has a synergistic effect in virus-receptor interactions.     

Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases.     

Degradation of human secretory IgA1 and IgA2 by Entamoeba histolytica surface-associated proteolytic activity

The protozoan Entamoeba histolytica is the etiological agent of amebiasis, an infection with high prevalence worldwide. The host-ameba relationship outcome depends on parasite and host factors, and among these is secretory IgA. These antibodies reduce mucosal colonization by pathogens and neutralize a variety of toxins and enzymes. The functionality of secretory IgA depends on its integrity. Some bacteria produce IgA proteases that cleave mainly the IgA1 subclass; live E. histolytica trophozoites, and other ameba fractions are also able to degrade human IgA. The aim of this study was to determine if serum and secretory IgA, its subclasses and secretory component, are degraded by cysteine proteases, which are present and active on the surface of glutaraldehyde-fixed amebas. It was observed that secretory IgA1, IgA2, free and IgA-bound secretory component were degraded by E. histolytica surface-associated cysteine proteinases. Secretory IgA2, although it was degraded, conserved its ability to agglutinate live amebas better than IgA1. Therefore, while specificity of known ameba cysteine proteases is cathepsin B-like and is different from bacterial IgA proteases, IgA2 was functionally more resistant than IgA1 to ameba surface-associated cysteine protease degradation, similar to the greater resistance of IgA2 to bacterial IgA-specific proteases.     

Cloning, expression, purification, and characterization of Streptococcus pneumoniae IgA1 protease

The IgA1 protease of Streptococcus pneumoniae is a Zn-metalloproteinase of 1964 amino acids that specifically cleaves the hinge region of IgA1, the predominant class of immunoglobulin present on mucosal membranes. This protease is associated to the bacterial cell surface via an N-terminal membrane anchor. Following proteolysis it is released in several forms of different molecular weight. Here, we describe the cloning, expression, and characterization of the enzymatic activity and immunogenicity of three fragments of IgA1 protease, including a large one lacking only the 103 N-terminal amino acids that constitute a typical prokaryotic signal sequence. Further, a proteolytically inactive mutant was generated by replacement of the glutamate residue with an alanine residue in the active site motif HExxH (1605-1609). This is the first report of recombinant active forms of S. pneumoniae IgA1 protease, which open the possibility of identifying specific inhibitors that could interfere with the mucosal colonization by pneumococcus. Moreover the inactive mutant could be considered as a candidate vaccine component.     

The Clostridium ramosum IgA proteinase represents a novel type of metalloendopeptidase

Clostridium ramosum is part of the normal flora in the human intestine. Some strains produce an IgA proteinase that specifically cleaves human IgA1 and the IgA2m(1) allotype. This prolylendopeptidase was purified from a broth culture supernatant, and N-terminal sequences of the native protein and tryptic fragments thereof were determined. A fragment of the iga gene encoding the IgA proteinase was isolated using degenerate primers in PCR, and the complete gene was obtained by inverse PCR. The identity of the iga gene was confirmed by heterologous expression in Escherichia coli. The deduced amino acid sequence indicated a signal peptide of 30 residues and a secreted proteinase of 133,828 Da. A typical Gram-positive cell wall anchor motif was identified in the C terminus. The presence of a putative zinc-binding motif His-Glu-Phe-Gly-His together with inhibition studies indicate that the proteinase belongs to the zinc-dependent metalloproteinases. However, the sequence of the C. ramosum IgA proteinase shows no overall similarity to other proteins except for significant identity around the zinc-binding motif with family M6 of metalloendopeptidases, and the unique sequence of the IgA proteinase in this area presumably establishes a new subfamily. The GC percentage of the iga gene is significantly higher than that for the entire genome of C. ramosum, suggesting that the gene was acquired recently in evolution.     

Extraintestinal pathogenic isolates of Escherichia coli do not possess active IgA1, IgA2, sIgA or IgG proteases.

Infections outside of the intestinal tract due to pathogenic strains of Escherichia coli result in significant morbidity, mortality and increased healthcare costs. The ability of these strains to cause both mucosal and systemic infections, as well as recurrent infections due to the same (homologous) strain suggests the hypothesis that strains of E. coli that cause infection outside of the intestinal tract possess proteases that are capable of cleaving IgA1, IgA2, sIgA or IgG. To test this hypothesis the ability of eight E. coli strains, isolated from sites outside of the urinary tract and 14 homologous and 11 heterologous strains of E. coli that were isolated from women with recurrent UTI, to cleave IgA1, IgA2, sIgA or IgG was evaluated. Our experimental design allowed for detection of cell-associated and secreted immunoglobulin proteases in both log and stationary phase. Surprisingly, none of these 33 human clinical isolates when grown in iron depleted Luria-Bertani medium or human urine were able to degrade the immunoglobulins assessed. Despite previous studies suggesting otherwise, the findings from this study support the concept that strains of E. coli that cause infection outside of the intestinal tract do not possess proteases that cleave the human immunoglobulins IgA1, IgA2, sIgA or IgG.     

G1m(1) and G1m(2) allotypes in Albanian towns of Calabria

The G1m(1) and G1m(2) allotype distribution was analyzed in a population sample from 11 Albanian towns of Calabria. The unusually high frequency of the G1m(1) marker already observed in Calabria as well as the presence of the Gm(2) phenotype were shown. The Calabrian and Albanian populations were similar, but significantly different from other Italian populations.