Diet and immunity have been known to be linked to each other for centuries. In the last 30 years systematic studies have confirmed that nutrient deficiencies impair immune response and lead to frequent severe infections resulting in increased mortality, especially in children. Protein-energy malnutrition results in reduced number and functions of T-cells, phagocytic cells and secretory immunoglobulin A antibody response. In addition, levels of many complement components are reduced. Similar findings have been reported for moderate deficiencies of individual nutrients such as trace minerals and vitamins, particularly Zn, Fe, Se, vitamins A, B6, C and E. For example, Zn deficiency is associated with profound impairment of cell-mediated immunity such as lymphocyte stimulation response, decreased CD4+:CD8+ cells, and decreased chemotaxis of phagocytes. In addition, the level of thymulin, which is a Zn-dependent hormone, is markedly decreased. The use of nutrient supplements, singly or in combination, stimulates immune response and may result in fewer infections, particularly in the elderly, low-birth-weight infants and malnourished critically-ill patients in hospitals. The interactions between nutrition and the immune system are of clinical, practical and public health importance.

As a member of the indigenous gut mucosal microbiota, segmented filamentous bacteria (SFB) colonize the guts of a variety of vertebrates and invertebrates. They are potent microbial stimuli of the gut mucosal immune system. In the small intestines of mice and rats, it has been observed that SFB are absent during the suckling period and appear in high numbers shortly after weaning, then quickly retreat to the cecum and large intestine. In this study, we explored whether this microecological phenomenon resulted from the interaction between SFB and the passively acquired maternal mucosal immunity and/or the actively acquired mucosal immunity. We set up a mouse model by reciprocal crossings and backcrossings of SFB-monoassociated, formerly germ-free, immunocompetent (+/+) BALB/c mice and immunodeficient (scid/scid) mice to produce pups which are either immunocompetent (scid/+) or immunodeficient (scid/scid) and are born either to immunocompetent (scid/+) mothers or to immunodeficient (scid/scid) mothers. We monitored the number of SFB on the mucosa of the small intestine in the four different groups of mice after birth, as well as the level of passively acquired antibodies, the active gut mucosal immune responses, and immunoglobulin A (IgA) coating of SFB in the gut. The results showed that, irrespective of whether the pups were scid/scid or scid/+, SFB could be found earlier on the mucosa of the small intestine in pups born to scid/scid mothers, appearing from day 13 and rapidly reaching a climax around weaning time on day 28, compared to the significantly delayed colonization in the pups of scid/+ mothers, starting from day 16 and peaking around days 28 to 32. After the climax, SFB quickly declined to very low levels in the small intestines of scid/+ pups of either scid/scid mothers or scid/+ mothers, whereas they remained at high levels in scid/scid pups at least until day 70, the last observation time in this study. The dynamic changes in SFB colonization of the small intestines of the different groups of pups may be related to the dynamic changes in the levels of SFB coated with secretory IgA (sIgA), which resulted from the significantly different levels of sIgA obtained from the mothers' milk during the suckling period and, later, of self-produced sIgA in the small intestine. Nevertheless, it is evident that the timing, localization, and persistence of colonization of the neonatal gut by SFB depends on the immune status of both mothers and pups.

Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnA(loxP/loxP) females with GATA1-Cre males. FlnA(loxP/y) GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbalpha because they lack the normal cytoskeletal linkage of GPIbalpha to underlying actin filaments. This results in approximately 70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-like-mediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased alpha-granule secretion, integrin alphaIIbbeta3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase C-gamma2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnA-Syk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-like-containing receptor signaling and platelet function.

Genetically modified derivatives of cholera toxin (CT), harboring a single amino acid substitution in and around the NAD binding cleft of the A subunit, were isolated following site-directed mutagenesis of the ctxA gene. Two mutants of CT, designated CTS106 (with a proline-to-serine change at position 106) and CTK63 (with a serine-to-lysine change at position 63), were found to have substantially reduced ADP-ribosyltransferase activity and toxicity; CTK63 was completely nontoxic in all assays, whereas CTS106 was 10(4) times less toxic than wild-type CT. The mucosal adjuvanticity and immunogenicity of derivatives of CT were assessed by intranasal immunization of mice, with either ovalbumin or fragment C of tetanus toxin as a bystander antigen. Mice immunized with wild-type CT produced both local (immunoglobulin A in mucosal washes) and systemic immune responses to both CT and bystander antigens. CTS106 showed good local and systemic responses to bystander proteins and to itself. Interestingly, mice immunized with the nontoxic derivative of CT, CTK63, generated weak immune responses to the bystander antigens which were similar to those achieved when CT B subunit was used as an adjuvant. In parallel experiments, an equivalent nontoxic mutant of the Escherichia coli heat-labile enterotoxin, LTK63 (with a serine-to-lysine change at position 63), was tested (9). In contrast to CTK63, LTK63 was found to be more immunogenic and a better intranasal adjuvant than recombinant heat-labile enterotoxin B subunit or CTK63. This information, together with data on immunoglobulin subclass responses, suggests that although highly homologous, CT and heat-labile enterotoxin should not be considered biologically identical in terms of their ability to act as intranasal adjuvants.

Liver, pancreas, and kidney from Pekin ducks infected with duck hepatitis B virus (DHBV) were assayed for the presence of both viral antigen and replication-specific forms of viral nucleic acid. In young congenitally infected ducks, antigen was detectable in hepatocytes and bile duct epithelia, in kidney glomeruli and tubular epithelia, and in cells localized to pancreatic acini. In older experimentally infected ducks, antigen was detectable in hepatocytes, in glomeruli and tubular epithelia, and in cells localized to presumptive pancreatic alpha-islets. All but the glomeruli-associated viral antigen appeared to be localized to the cytoplasm of antigen-positive cells. Much of the glomeruli-associated antigen appeared to be extracellular and was detected in glomeruli that were positive for the accumulation of immunoglobulin, observations suggestive of the deposition of viral antigen-antibody complexes. As analyzed with bulk tissue, replication-specific forms of viral nucleic acid were detectable in liver and pancreas from the young congenitally infected ducks and in liver and kidney from the older experimentally infected ducks.

The epithelial polymeric immunoglobulin receptor/transmembrane secretory component (pIgR/SC) transports into secretions polymeric immunoglobulin A (pIgA), which is considered the first line of defense of the respiratory tract. The present study, done with quantitative immunohistochemistry, evaluated epithelial expression of secretory component (SC) and Clara cell protein (CC16) and neutrophil infiltration into the airways of eight patients with severe chronic obstructive pulmonary disease (COPD) who were undergoing lung transplantation, as compared with these processes in six nonsmoking patients with pulmonary hypertension who were used as controls and in lung specimens from five smokers without chronic bronchitis. Staining for SC was significantly decreased in the COPD patients as compared with the controls, both in large (mean optical density [MOD]: 23.4 [range: 21.1 to 27.8] versus 42.2 [range: 28.2 to 49.3], p = 0.003) and in small airways (MOD: 30.8 [range: 20.3 to 39.4] versus 41.5 [range: 39.2 to 46.2], p = 0.003). SC expression in small airways correlated strongly with functional parameters such as FEV1 (Kendall's tau (K) = 0.76, p = 0.008), FVC (K = 0.64, p = 0.03), and midexpiratory flow at 50% of VC (MEF50) (K = 0.74, p = 0.01). The reduced expression of SC in large airways correlated with neutrophil infiltration in submucosal glands (K = -0.47, p = 0.03). Expression of CC16 in the bronchial epithelium of COPD patients was also significantly decreased as compared with that of controls, especially in small airways (MOD: 28.3 [range: 26.8 to 32.4] versus 45.8 [range: 40.7 to 56.0], p = 0.002), but no correlation was observed with lung function tests. In conclusion, this study shows that reduced expression of SC in airway epithelium is associated with airflow obstruction and neutrophil infiltration in severe COPD.

There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining immunoglobulin A (IgA) transcytosis across Transwells. IgA transcytosis required induction of polymeric Ig receptor (pIgR) expression, which could be stimulated by a combination of lipopolysaccharide and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that tumor necrosis factor-α, interleukin (IL)-1β, IL-17, and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that among these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. Interferon-γ, however, did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology.

The ability of mucosally delivered plasmid DNA encoding glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1) to generate systemic as well as distal mucosal immunity was evaluated. BALB/c mice were immunized intranasally (i.n.) with gB DNA or DNA expressing beta-galactosidase (beta-Gal). Two days following immunization, gB and beta-Gal gene expression was detected by reverse transcription (RT)-PCR in lungs and cervical lymph nodes (CLN). Histological analysis showed that beta-Gal protein was expressed in vivo in the lungs and the CLN of animals immunized with i.n. administered beta-Gal DNA. The immune responses generated by i.n. administration of gB DNA with or without cholera toxin (CT) were compared to those generated by intramuscular (i.m.) gB DNA and i.n. live HSV administration. Three i.n. doses of gB DNA over a 3-week period resulted in a distal mucosal immunoglobulin A (IgA) response. In addition, the mucosal IgA response was enhanced by coadministration of CT with gB DNA. The i.m. route of immunization induced a strong IgG response in the serum and vagina but was inefficient in generating a mucosal IgA response. Antigen-specific cytokine ELISPOT analyses as well as the serum IgG1/IgG2a ratio indicated induction of stronger Th2 responses following the additional i.n. administration of CT compared to i.n. or i.m. gB DNA or i.n. live HSV immunization. In addition, mucosal immunization with gB DNA induced anti-HSV cell-mediated immunity in vivo as measured by delayed-type hypersensitivity. Although i.n. DNA immunization was an effective means of inducing mucosal antibody, it was inferior to i.m. DNA delivery in providing protection against lethal HSV challenge via the vaginal route. In addition, both i.m. and i.n. plasmid immunizations failed to generate an immune barrier to viral invasion of the mucosa.

OBJECTIVE

HIV-neutralizing immunoglobulin A (IgA) and HIV-specific cellular immunity have been described in highly exposed, persistently seronegative (HEPS) individuals, but well controlled studies have not been performed. We performed a prospective, nested case-control study to examine the association of genital IgA and systemic cellular immune responses with subsequent HIV acquisition in high-risk Kenyan female sex workers (FSWs).

METHODS

A randomized trial of monthly antibiotic prophylaxis to prevent sexually transmitted disease/HIV infection was performed from 1998 to 2002 in HIV-uninfected Kenyan FSWs. After the completion of trial, FSWs who had acquired HIV (cases) were matched 1: 4 with persistently uninfected controls based on study arm, duration of HIV-seronegative follow-up, and time of cohort enrolment. Blinded investigators assayed the ability at enrolment of genital IgA to neutralize primary HIV isolates as well as systemic HIV-specific cellular IFNgamma-modified enzyme-linked immunospot and proliferative responses.

RESULTS

The study cohort comprised 113 FSWs: 24 cases who acquired HIV and 89 matched controls. Genital HIV-neutralizing IgA was associated with reduced HIV acquisition (P = 0.003), as was HIV-specific proliferation (P = 0.002), and these associations were additive. HIV-specific IFNgamma production did not differ between case and control groups. In multivariable analysis, HIV-neutralizing IgA and HIV-specific proliferation each remained independently associated with lack of HIV acquisition. Genital herpes (HSV2) was associated with increased HIV risk and with reduced detection of HIV-neutralizing IgA.

CONCLUSIONS

Genital HIV-neutralizing IgA and systemic HIV-specific proliferative responses, assayed by blinded investigators, were prospectively associated with HIV nonacquisition. The induction of these immune responses may be an important goal for HIV vaccines.

Both in vitro and in vivo studies established that interleukin 7 (IL-7) is essential for differentiation of immature T cells and B cells but not natural killer (NK) cells in the mouse. In humans, although both T-cell and B-cell progenitors express the functional IL-7 receptor that consists of IL-7R alpha and the gamma common (gamma c) chain, this lymphocyte receptor system is critical for T lineage but not for B lineage development. Indeed, complete gamma c deficiency like IL-7R alpha deficiency results in the arrest of T-cell but not B-cell development (T(-)B(+) SCID). However, partial deficiency of gamma c caused by missense mutations results in a T(+)B(+) phenotype and a delay of clinical presentation. It was therefore plausible to assume that partial deficiency of IL-7R alpha, like partial gamma c deficiency may lead to a milder clinical and immunologic phenotype. A P132S mutation in the IL-7R alpha was identified in 3 patients with severe combined immunodeficiency (SCID) within an extensively consanguineous family. Substitution of proline with serine in the extracellular portion of IL-7R alpha did not affect IL-7R alpha messenger RNA (mRNA) and protein expression, but severely compromised affinity to IL-7, resulting in defective signal transduction. In response to IL-7 stimulation, Jak-3 phosphorylation was markedly reduced in both patient cells as well as in COS cells reconstituted with mutant IL-7R alpha. Surprisingly, this partial deficiency of IL-7R alpha resulted in a severe phenotype, including markedly reduced circulating T cells while sparing B-cell numbers similar to gamma c chain deficiency. However, unlike the previously reported cases, serum immunoglobulins were virtually absent. Further, unlike gamma c deficiency, NK cell numbers and function was preserved. Despite the partial deficiency, clinical presentation was indistinguishable from a complete gamma c deficiency, including severe and persistent viral and protozoal infections and failure to thrive. Unlike partial gamma c deficiency, a partial deficiency of IL-7R alpha results in an arrest of T-cell development, leading to typical severe combined immunodeficiency. This underscores the critical role of IL-7R alpha chain in the differentiation of T cells. (Blood. 2000;96:2803-2807)

Intestinal epithelial cells (IEC) play a central role in innate and acquired mucosal immunity. They ensure early signaling to trigger an inflammatory response against pathogens. Moreover, IEC mediate transcytosis of dimeric IgA (dIgA), through the polymeric-immunoglobulin receptor (pIgR), to provide secretory IgA, the major protective Ig in mucosal secretions. Using an in vitro model of polarized IEC, we describe an additional anti-inflammatory mechanism of dIgA-mediated protection against intracellular bacterial components involved in the proinflammatory activation of IEC. Specific dIgA colocalizes to lipopolysaccharide (LPS) in the apical recycling endosome compartment, preventing LPS-induced NF-kappaB translocation and subsequent proinflammatory response. Thus, intracellular neutralization by dIgA limits the acute local inflammation induced by proinflammatory pathogen-associated molecular patterns such as LPS.

Mature human milk contains 3%--5% fat, 0.8%--0.9% protein, 6.9%--7.2% carbohydrate calculated as lactose, and 0.2% mineral constituents expressed as ash. Its energy content is 60--75 kcal/100 ml. Protein content is markedly higher and carbohydrate content lower in colostrum than in mature milk. Fat content does not vary consistently during lactation but exhibits large diurnal variations and increases during the course of each nursing. Race, age, parity, or diet do not greatly affect milk composition and there is no consistent compositional difference between milks from the two breasts unless one is infected. The principal proteins of human milk are a casein homologous to bovine beta-casein, alpha-lactalbumin, lactoferrin, immunoglobulin IgA, lysozyme, and serum albumin. Many enzymes and several "minor" proteins also occur. The essential amino acid pattern of human milk closely resembles that found to be optimal for human infants. Possible special functions of milk proteins and enzymes other than as a source of amino acids, are as yet largely speculative. The principal sugar of human milk is lactose but 30 or more oligosaccharides, all containing terminal Gal-(beta 1,4)-Glc and ranging from 3--14 saccharide units per molecule are also present. These may amount in the aggregate to as much as 1 g/100 ml in mature milk and 2.5 g/100 ml in colostrum. Some of them may function to control intestinal flora because of their ability to promote growth of certain strains of lactobacilli. Human milk fat is characterized by high contents of palmitic and oleic acids. the former heavily concentrated in the 2-position and the latter in the 1- and 3-positions of the triglycerides. Fatty acid composition of milk fat varies somewhat with the composition of diet, particularly the fatty acids which it supplies. Phospholipids, amounting in the aggregate to about 75 mg/100 ml, include phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, and sphingomyelin. The principal mineral constituents of human milk are Na, K, Ca, Mg, P, and Cl. Calcium concentrations reported in various studies vary from 25--35 mg/100 ml. Phosphorus at 13--16 mg/100 ml is much more constant but is lower in proportion to casein and calcium than in milks of most other species. Iron, copper, and zinc contents of human milk vary considerably. A long list of other trace elements has been reported. About 25% of the total nitrogen of human milk represents nonprotein compounds including urea, uric acid, creatine, creatinine, and a large number of amino acids. Of the latter, glutamic acid and taurine are prominent. All of the vitamins, except K, are found in human milk in nutritionally significant concentrations.

Five gram-positive bacterial strains were selected for absorption studies of human serum samples. Strain AR1 (group A, M-type 1) and G148 (group G), with strong immunoglobulin G (IgG) binding capacities, and strain AW43 (group A, M-type 60), binding both IgA1 and IgA2, were compared with Staphylococcus aureus Cowan I and with Staphylococcus epidermidis L603. Both AR1 and G148 were capable of completely absorbing out serum IgG. In contrast, S. aureus Cowan I left a fraction unabsorbed, as expected from its known lack of IgG3 binding. Strain AW43 absorbed out all serum IgA, using a 10-microliter bacterial pellet for 20 microliter of serum. Serum IgM levels were slightly reduced by S. aureus Cowan I absorption. On the basis of the experiments, a bacterial mixture was designed consisting of S. aureus Cowan I and group A streptococcus strains AR1 and AW43, with absorption characteristics suitable for use in discriminating between early IgM and late IgG and IgA immune responses in routine serological work. A new type of bacteria-mammalian protein binding was discovered. Human serum albumin was completely absorbed out by strain G148 and to a lesser extent by strain AR1 and AW43. S. aureus Cowan I and S. epidermidis were negative. The binding capacity of G148 for albumin equalled that of Cowan I for IgG. The binding pattern of albumin to the strains was different from those of IgG, IgA, IgM, fibrinogen, haptoglobin, or aggregated beta 2-microglobulin and therefore seems to represent another type of bacterial-mammalian interaction with a specific albumin receptor on the surface of streptococci.

We explored the immunogenic properties of influenza A viruses with altered NS1 genes (NS1 mutant viruses). NS1 mutant viruses expressing NS1 proteins with an impaired RNA-binding function or insertion of a longer foreign sequence did not replicate in murine lungs but still were capable of inducing a Th1-type immune response resulting in significant titers of virus-specific serum and mucosal immunoglobulin G2 (IgG2) and IgA, but with lower titers of IgG1. In contrast, replicating viruses elicited high titers of serum and mucosal IgG1 but less serum IgA. Replication-deficient NS1 mutant viruses induced a rapid local release of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Moreover, these viruses also elicited markedly higher levels of IFN-alpha/beta in serum than the wild-type virus. Comparable numbers of virus-specific primary CD8(+) T cells were determined in all of the groups of immunized mice. The most rapid onset of the recall CD8(+)-T-cell response upon the wild-type virus challenge was detected in mice primed with NS1 mutant viruses eliciting high levels of cytokines. It is noteworthy that there was one NS1 mutant virus encoding NS1 protein with a deletion of 40 amino acids predominantly in the RNA-binding domain that induced the highest levels of IFN-alpha/beta, IL-6 and IL-1beta after infection. Mice that were immunized with this virus were completely protected from the challenge infection. These findings indicate that a targeted modification of the RNA-binding domain of the NS1 protein is a valuable technique to generate replication-deficient, but immunogenic influenza virus vaccines.

Carriage of the natural killer (NK) receptor genotype KIR3DL1*h/*y with its HLA-B*57 ligand (*h/*y+B*57) is associated with slow time to AIDS and low viral load (VL). To provide a functional basis for these epidemiological observations, we assessed whether HIV-1-infected slow progressors (SP) carrying the *h/*y+B*57 compound genotype would have increased NK cell polyfunctional potential in comparison to SP with other killer immunoglobulin-like receptor (KIR)/HLA compound genotypes and whether this enhanced polyfunctionality was dependent upon the coexpression of both KIR3DL1*h/*y and HLA-B*57. The functional potential of NK cells was investigated by stimulating peripheral blood mononuclear cells with HLA-devoid targets or single HLA transfectants. Multiparametric flow cytometry was used to detect NK cells with seven functional profiles representing all permutations of CD107a expression and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion. NK cells from individuals carrying KIR3DL1 receptor-HLA-Bw4 ligand pairs had greater trifunctional responses than those from KIR3DL1 homozygotes (hmz), who were Bw6 homozygotes. NK cells from subjects carrying the *h/*y+B*57 genotypes exhibited the highest trifunctional potential, and this was dependent on cocarriage of the NK receptor and its ligand. Trifunctional cells secreted more of each function tested on a per-cell basis than each corresponding monofunctional NK subset. Although VL influenced NK functionality, individuals with defined KIR/HLA genotypes exhibited differences in NK cell polyfunctionality that could not be accounted for by VL alone. The protective effect of HLA-B*57 on slow progression to AIDS and low VL may be mediated through its interaction with KIR3DL1 alleles to educate NK cells for potent activity upon stimulation.

The lipopolysaccharide (LPS) of gram-negative bacteria serves as a barrier between the cell and its environment. The LPS O antigen is the immunodominant portion of the molecule and thus has a significant effect on the interaction between a bacterial pathogen and the host organism. Antibodies directed against O antigen are vital to the immune response to infection. In this study, we have characterized the interaction between a series of monoclonal immunoglobulin A antibodies and the LPS of Salmonella typhimurium. Using one of these antibodies, we have previously shown that monoclonal immunoglobulin A is sufficient to protect against S. typhimurium infection, both in vivo and in vitro. Here, we show that recognition of LPS by the monoclonal antibodies is affected by acetylation of the O antigen on the abequose moiety, the determinant of the O5 epitope. Although recognition of LPS by several of the monoclonal antibodies is completely dependent on acetylation, the antibodies recognize clearly separable epitopes. This suggests that acetylation of O antigen affects the three-dimensional structure of the molecule and thus creates and destroys a series of conformational antigenic determinants. We have shown that a change in the acetylation state of LPS has no effect on virulence. However, acetylation has important consequences for the mucosal immune response and thus could potentially have profound implications for the ability of an immune host to respond to a subsequent infection.

This study aimed at investigating the fecal microbiota, and the fecal and urinary metabolome of non progressor (NP) and progressor (P) patients with immunoglobulin A nephropathy (IgAN). Three groups of volunteers were included in the study: (i) sixteen IgAN NP patients; (ii) sixteen IgAN P patients; and (iii) sixteen healthy control (HC) subjects, without known diseases. Selective media were used to determine the main cultivable bacterial groups. Bacterial tag-encoded FLX-titanium amplicon pyrosequencing of the 16S rDNA and 16S rRNA was carried out to determine total and metabolically active bacteria, respectively. Biochrom 30 series amino acid analyzer and gas-chromatography mass spectrometry/solid-phase microextraction (GC-MS/SPME) analyses were mainly carried out for metabolomic analyses. As estimated by rarefaction, Chao and Shannon diversity index, the lowest microbial diversity was found in P patients. Firmicutes increased in the fecal samples of NP and, especially, P patients due to the higher percentages of some genera/species of Ruminococcaceae, Lachnospiraceae, Eubacteriaceae and Streptococcaeae. With a few exceptions, species of Clostridium, Enterococcus and Lactobacillus genera were found at the highest levels in HC. Bacteroidaceae, Porphyromonadaceae, Prevotellaceae and Rikenellaceae families differed among NP, P and HC subjects. Sutterellaceae and Enterobacteriaceae species were almost the highest in the fecal samples of NP and/or P patients. Compared to HC subjects, Bifidobacterium species decreased in the fecal samples of NP and P. As shown by multivariate statistical analyses, the levels of metabolites (free amino acids and organic volatile compounds) from fecal and urinary samples markedly differentiated NP and, especially, P patients.

Immunoglobulin A (IgA), the major class of antibody secreted by the gut mucosa, is an important contributor to gut barrier function. The repertoire of IgA bound to gut bacteria reflects both T-cell-dependent and -independent pathways, plus glycans present on the antibody's secretory component. Human gut bacterial taxa targeted by IgA in the setting of barrier dysfunction are capable of producing intestinal pathology when isolated and transferred to gnotobiotic mice. A complex reorientation of gut immunity occurs as infants transition from passively acquired IgA present in breast milk to host-derived IgA. How IgA responses co-develop with assembly of the microbiota during this period remains poorly understood. Here, we (1) identify a set of age-discriminatory bacterial taxa whose representations define a program of microbiota assembly and maturation during the first 2 postnatal years that is shared across 40 healthy twin pairs in the USA; (2) describe a pattern of progression of gut mucosal IgA responses to bacterial members of the microbiota that is highly distinctive for family members (twin pairs) during the first several postnatal months then generalizes across pairs in the second year; and (3) assess the effects of zygosity, birth mode, and breast feeding. Age-associated differences in these IgA responses can be recapitulated in young germ-free mice, colonized with faecal microbiota obtained from two twin pairs at 6 and 18 months of age, and fed a sequence of human diets that simulate the transition from milk feeding to complementary foods. Most of these responses were robust to diet, suggesting that 'intrinsic' properties of community members play a dominant role in dictating IgA responses. The approach described can be used to define gut mucosal immune development in health and disease states and to help discover ways of repairing or preventing perturbations in this facet of host immunity.

In testing the hypothesis that interleukin-4 receptor alpha-subunit (IL-4R alpha)-coupled signaling mediates altered airway smooth muscle (ASM) responsiveness in the atopic sensitized state, isolated rabbit tracheal ASM segments were passively sensitized with immunoglobulin E (IgE) immune complexes, both in the absence and presence of an IL-4R alpha blocking antibody (anti-IL-4R alpha Ab). Relative to control ASM, IgE-sensitized tissues exhibited enhanced isometric constrictor responses to administered ACh and attenuated relaxation responses to isoproterenol. These proasthmatic-like effects were prevented in IgE-sensitized ASM that were pretreated with anti-IL-4R alpha Ab. In complementary experiments, IgE-sensitized cultured human ASM cells exhibited upregulated expression of IL-13 mRNA and protein, whereas IL-4 expression was undetected. Moreover, extended studies demonstrated that 1) exogenous IL-13 administration to naïve ASM elicited augmented contractility to ACh and impaired relaxation to isoproterenol, 2) these effects of IL-13 were prevented by pretreating the tissues with an IL-5 receptor blocking antibody, and 3) IL-13 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of IgE-sensitized ASM is largely attributed to activation of an intrinsic Th2-type autocrine mechanism involving IL-13/IL-4R alpha-coupled release and action of IL-5 in the sensitized ASM itself.

A novel arginine-specific cysteine proteinase, termed "argingipain," was purified from culture supernatants of Porphyromonas gingivalis, an anaerobe commonly associated with progressive periodontal disease, by conventional chromatographic techniques. The purified enzyme was found to be composed of a single polypeptide of M(r) approximately 44,000. Analysis of the enzymatic properties revealed several distinctive features for this enzyme. The proteolytic activity is absolutely thiol-dependent, but the enzyme also has in part the characteristics of both metallo and serine endopeptidases, as shown by the inhibition of activity by metal chelators, chymostatin, and the chloromethyl ketones of tosyl-L-lysine and tosyl-L-phenylalanine. However, internal protease inhibitors, such as cystatins, tissue inhibitor of metalloproteinases, and alpha 1-antichymotrypsin, have no effects on the activity, suggesting its evasion from normal host defense systems in vivo. Despite its narrow specificity for synthetic substrates containing Arg in the P1 site and hydrophobic amino acids in the P2 or P3 sites, the enzyme extensively degrades collagens (types I and IV) and immunoglobulin G. Most important, the enzyme has the ability to disrupt the functions of polymorphonuclear leukocytes, as shown by its inhibitory effect on the generation of active oxygen species from the activated cells. Further, the enzyme is found to be produced by all of the species of P. gingivalis examined, but not by other bacteria. These results suggests that argingipain plays a key role as a major virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction of periodontal tissue components and the disruption of normal host defense mechanisms.

Human colostrum, parotid saliva, and serum were assayed for the presence of naturally occurring antibodies to five serotypes of Streptococcus mutans. Appreciable levels of agglutinins to strains AHT, BHT, 10449, 6715, and LM-7 (groups a leads to e, respectively) were detected in normal colostrum and saliva, whereas relatively low levels were found in serum. No agglutinins could be detected in the colostrum or saliva of immunodeficient patients. Molecular sieve chromatography of the colostrum on Sephadex G-200 revealed agglutinin activity in the secretory immunoglobulin A (s-IgA)-rich fraction only. Titration of purified colostral s-IgA confirmed the IgA nature of this agglutinating activity. Indirect immunofluorescence tests with anti-s-IgA, -IgG, and -IgM revealed S. mutans specificity only in the s-IgA class. The presence of s-IgA antibodies to indigenous oral microorganisms in colostrum, as well as in saliva, suggests that antigenic stimulation occurs at a site remote from the oral mucosa.

We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cell line that carries a reciprocal translocation between chromosomes 8 and 14. The breakpoint lies within the first intron of c-myc, so that the first noncoding exon of the gene remains on the 8q- chromosome. The second and third coding exons are translocated to the 14q+ chromosome into the switch region of C-alpha 1. The orientation of the c-myc gene with relationship to alpha 1 is 5' to 5', with directions of transcription in opposite orientation. DNA sequencing studies predict five changes in the amino acid sequence of the myc protein, two of which occur in a region within the second exon which is highly conserved in evolution. Southern blotting data indicate that the first exon of c-myc is rearranged 3' to 3' with the pseudo-epsilon gene. Because CA46 cells contain two rearranged mu genes, the translocation must have occurred after immunoglobulin rearrangement. The position of the breakpoint in CA46 occurs within a 20-base-pair region of the first intron of c-myc to which breakpoints have been mapped for two additional B-cell lymphomas with the t(8;14) translocation, ST486 and the Manca cell line. The region of the heavy chain locus to which c-myc has translocated is different in each case. Comparisons have been made of the levels of transcripts of the translocated c-myc gene in ST486 and CA46, where the gene is not associated with the heavy chain enhancer, with its expression in the Manca cell, in which it is. The c-myc gene is transcribed at similar levels in all three cases.

C57BL/6 mice were orally immunized with five weekly doses of 2 mg, 200 microgram, or 2 microgram of Helicobacter pylori (Sydney strain) whole-cell sonicate combined with cholera toxin. One week after the last vaccination, mice were challenged with 5 x 10(7) CFU of live H. pylori three times at 2-day intervals. At 6 or 18 weeks after the challenge, mice were sacrificed and bacterial cultures and histological studies of the stomach were performed. Vaccination with 2 mg/session or 200 microgram/session inhibited H. pylori colonization by 90 and 100%, respectively. These mice were considered protected. Lower levels of H. pylori-specific immunoglobulin A (IgA) were detected in fecal and saliva samples before challenge. However, a significant increase in IgA secretion in mucosal tissue and a higher labeling index for IgA-positive lumina of pyloric glands were noted in these mice in response to challenge and in a vaccine dose-dependent manner. In protected mice, however, severe gastritis characterized by marked infiltration of inflammation mononuclear cells was noted at 6 weeks after challenge, compared with the gastritis seen in unprotected mice or nonvaccinated, ordinarily infected mice. Marked expression of gamma interferon mRNA was detected in the stomach of all protected mice, and 50% of these mice expressed interleukin 4 (IL-4) or IL-5 mRNA. Our findings suggest that local secretory IgA antibody and severe postimmunization gastritis correlate well with protection of mice against H. pylori infection.