抗核抗体核型分布及核型与特异性抗体相关性分析

目的通过对ANA核型分布及核型与特异性抗体相关性分析,为临床对ANA和ANA谱检测结果判读提供参考。方法用间接免疫荧光法检测ANA,免疫印迹法检测ANA谱,对449例ANA或ANA谱阳性的结果进行核型和特异性抗体分析。结果 ANA核型分布以核颗粒型为主,占78.2%;核型与特异性抗体有一定的相关性,但同一种自身抗体可出现不同的荧光核型。结论 ANA和ANA谱同时检测能提高AID的检出率,有助于准确的判断特异性抗体,对AID的诊断、病情监测、预后判断有十分重要的作用。     

Magnitude of Serum and Intestinal Antibody Responses Induced by Sequential Replicating and Nonreplicating Rotavirus Vaccines in Gnotobiotic Pigs and Correlation with Protection

A sequential mucosal prime-boost vaccine regimen of oral attenuated (Att) human rotavirus (HRV) priming followed by intranasal (i.n.) boosting with rotavirus protein VP2 and VP6 rotavirus-like particles (2/6-VLPs) has previously been shown to be effective for induction of intestinal antibody-secreting cell (ASC) responses and protection in gnotobiotic pigs. Because serum or fecal antibody titers, but not intestinal ASC responses, can be used as potential markers of protective immunity in clinical vaccine trials, we determined the serum and intestinal antibody responses to this prime-boost rotavirus vaccine regimen and the correlations with protection. Gnotobiotic pigs were vaccinated with one of the two sequential vaccines: AttHRV orally preceding 2/6-VLP (VLP2x) vaccination (AttHRV/VLP2x) or following VLP2x vaccination (VLP2x/AttHRV) given i.n. with a mutant Escherichia coli heat-labile toxin (mLT) as adjuvant. These vaccines were also compared with three i.n. doses of VLP+mLT (VLP3x) and one and three oral doses of AttHRV (AttHRV1x and AttHRV3x, respectively). Before challenge all pigs in the AttHRV/VLP2x group seroconverted to positivity for serum immunoglobulin A (IgA) antibodies. The pigs in this group also had significantly higher (P < 0.05) intestinal IgA antibody titers pre- and postchallenge and IgG antibody titers postchallenge compared to those in the other groups. Statistical analyses of the correlations between serum IgM, IgA, IgG, and virus-neutralizing antibody titers and protection demonstrated that each of these was an indicator of protective immunity induced by the AttHRV3x and the AttHRV/VLP2x regimens. However, only IgA and not IgM or IgG antibody titers in serum were highly correlated (R² = 0.89; P < 0.001) with the corresponding isotype antibody (IgA) titers in the intestines among all the vaccinated groups, indicating that the IgA antibody titer is probably the most reliable indicator of protection.     

Different Macrophage Requirement in the Specific and Polyclonal Responses Induced by TNP-LPS and LPS

The in vitro anti-trinitrophenyl (TNP) response induced by TNP-lipopolysaccharide (TNP-LPS) in mouse spleen cells was eliminated by passage through Sephadex G10 columns. Conditioned medium obtained from peritoneal macrophages restored the response, indicating the supportive role of such cells in the response. Conversely, polyclonal B-cell activation mediated by LPS was not affected by Sephadex G10 treatment, as judged by incorporation of ³H-thymidine, generation of specific anti-TNP plaque-forming cells, and induction of polyclonal IgM-secreting cells. The failure of the LPS moiety of TNP-LPS to induce B-cell activation without macrophage help suggests a restriction in the number of available anti-LPS receptor molecules when the Ig anti-TNP receptor interacts with the haptenic (TNP) groups. This restriction can be due to the attachment of the TNP molecules to residues located in the vicinity of the lipid A mitogenic region of the carrier.     

SARS-CoV-2 Vaccine Induced Atypical Immune Responses in Antibody Defects: Everybody Does their Best

Background

Data on immune responses to SARS-CoV-2 in patients with Primary Antibody Deficiencies (PAD) are limited to infected patients and to heterogeneous cohorts after immunization.

Methods

Forty-one patients with Common Variable Immune Deficiencies (CVID), six patients with X-linked Agammaglobulinemia (XLA), and 28 healthy age-matched controls (HD) were analyzed for anti-Spike and anti-receptor binding domain (RBD) antibody production, generation of Spike-specific memory B-cells, and Spike-specific T-cells before vaccination and one week after the second dose of BNT162b2 vaccine.

Results

The vaccine induced Spike-specific IgG and IgA antibody responses in all HD and in 20% of SARS-CoV-2 naive CVID patients. Anti-Spike IgG were detectable before vaccination in 4 out 7 CVID previously infected with SARS-CoV-2 and were boosted in six out of seven patients by the subsequent immunization raising higher levels than patients naïve to infection. While HD generated Spike-specific memory B-cells, and RBD-specific B-cells, CVID generated Spike-specific atypical B-cells, while RBD-specific B-cells were undetectable in all patients, indicating the incapability to generate this new specificity. Specific T-cell responses were evident in all HD and defective in 30% of CVID. All but one patient with XLA responded by specific T-cell only.

Conclusion

In PAD patients, early atypical immune responses after BNT162b2 immunization occurred, possibly by extra-follicular or incomplete germinal center reactions. If these responses to vaccination might result in a partial protection from infection or reinfection is now unknown. Our data suggests that SARS-CoV-2 infection more effectively primes the immune response than the immunization alone, possibly suggesting the need for a third vaccine dose for patients not previously infected.

     

Correlation between Specific Immunosuppression and Polyclonal B Cell Activation Induced by a Protein Secreted by Streptococcus mutans

The relationship between polyclonal B cell activation and immunosuppressor effects induced by F5'EP-Sm, a non-cytotoxic protein secreted by Streptococcus mutans , was studied in C57BL/6 mice. Mice created with F5'EP-Sm exhibited a considerable increase in splenic nonspecific Ig plaque-forming cells (PFC) compared with untreated mice. The isotypic pattern of non-specific PFC responses favours IgG2a∼IgG2b>IgG3>IgG1∼IgM, when taken as a ratio between treated and untreated animals. When F5'EP-Sm was administered 2 days before immunization with sheep red blood cells (SRBC), the non-specific PFC production was accompanied by an ephemeral increase in specific PFC against SRBC 1 day after immunization, which was quickly replaced by a strong immunosuppression. In contrast, when F5'EP-Sm was injected after priming, there was little or no demonstrable suppression of specific PFC, and the increase of non-specific PFC was much less evident. The kinetic curves representing increase or decrease in relation to controls of specific and non-specific PFC are almost mirror images in each of the isotypes. The in vivo suppressor effect was abrogated in thymectomized mice, although the involvement of the T cell compartment is probably secondary to the B cell mitogen effect, since T-depleted spleen cells proliferate and synthesize non-specific Ig when stimulated in vitro with F5'EP-Sm.     

Evaluation of Serum Antibody Responses against the Rotavirus Nonstructural Protein NSP4 in Children after Rhesus Rotavirus Tetravalent Vaccination or Natural Infection

The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S-transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly (P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of ≤200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant (P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate.     

Vaccine‐Induced Antibody Responses Prevent the Induction of Interferon Type I Responses Upon a Homotypic Live Virus Challenge

During acute viral infections, innate immunity provides essential protective measures to minimize virus dissemination and regulate adaptive immunity. This helps to successfully eliminate the pathogen and establish long‐term memory. Here, we investigated the effect of vaccine‐induced antibody responses on the induction of IFN‐I responses and the associated lymphocyte activation using influenza A virus vaccination and challenge models. Mice were vaccinated with gamma‐irradiated influenza A virus (γ‐FLU) and challenged three weeks later with live virus. Our data show a significant reduction in IFN‐I responses and lymphocyte activation following a homotypic virus challenge. We confirmed the role of vaccine‐induced antibody responses in the observed impairment of IFN‐I and the associated lymphocyte activation using adoptive transfer of immune sera and the administration of sera‐treated viruses prior to challenge. Overall, we addressed a fundamental concept in immunology and provided experimental data illustrating the inhibition of IFN‐I responses in vaccinated animals upon a homotypic virus challenge.     

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

Infectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses.     

Differences in Specific Antibody Responses of Human Tonsillar Cells to an Oral and a Parenteral Antigen

Tonsillar lymphocytes stimulated in vitro with either β-lactoglobulin or with tetanus toxoid were shown to produce specific antibodies by a direct plaque assay and by radioimmuno-precipitation of the culture supernatants. There was a sixfold increase in the number of IgA-secreting cells in response to β-lactoglobulin; no such effect was seen in response to tetanus toxoid, where a fivefold rise in IgG-secreting cells occurred. These differences in antibody response are probably due to the route of initial antigen presentation. Those antigens priming the mucosa-associated lymphoid system stimulate mainly IgA-producing cells, in contrast to parenteral antigens, which elicit a predominantly IgM and IgG response.     

Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Specific IgG Antibody and DTH Responses in Mice

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed-type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM-OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.     

Functional Differences in the Specific B-Cell Compartment in High or Low Antibody Responder Mice

The role of antigen-presenting cells (APC) in quantitative antibody synthesis regulation was studied in mice genetically selected for high (HI) or low (LI) antibody response. Irradiated spleen cells and enriched specific B cells from HI and LI mice co-isogenic at H-2 ^s locus, were compared for their capacity to present chicken ovalbumin (OVA) to specific T-cell hybridomas. Minor differences were observed between HI and LI mice when three distinct hybridomas were stimulated in the presence of OVA and splenic macrophages APC. These differences were totally abolished when APC were pulsed with OVAxAb complexes. Looking at the B-cell compartment, hybridoma IL-2 responses were similar when TNP primed B cells were pulsed with OVA. However, when OVA was targeted on TNP-specific enriched B cells by pulsing with TNP-OVA, the IL-2 production by the T-cell hybridomas was stronger in the presence of HI B cells than in the presence of LI B cells. These results strongly suggest that an efficient Ag handling/processing by specific B cells is a major component of the high Ab responder status in Biozzi mice.     

Functional Differences in the Specific B-Cell Compartment in High or Low Antibody Responder Mice

The role of antigen-presenting cells (APC) in quantitative antibody synthesis regulation was studied in mice genetically selected for high (HI) or low (LI) antibody response. Irradiated spleen cells and enriched specific B cells from HI and LI mice co-isogenic at H-2 ^s locus, were compared for their capacity to present chicken ovalbumin (OVA) to specific T-cell hybridomas. Minor differences were observed between HI and LI mice when three distinct hybridomas were stimulated in the presence of OVA and splenic macrophages APC. These differences were totally abolished when APC were pulsed with OVAxAb complexes. Looking at the B-cell compartment, hybridoma IL-2 responses were similar when TNP primed B cells were pulsed with OVA. However, when OVA was targeted on TNP-specific enriched B cells by pulsing with TNP-OVA, the IL-2 production by the T-cell hybridomas was stronger in the presence of HI B cells than in the presence of LI B cells. These results strongly suggest that an efficient Ag handling/processing by specific B cells is a major component of the high Ab responder status in Biozzi mice.     

Natural Killing of Herpes Simplex Virus Type 1-Infected Target Cells: Normal Human Responses and Influence of Antiviral Antibody

Studies of a mouse model of genetic resistance to herpes simplex virus type 1 (HSV-1) indicate that the marrow-dependent effector cell of allogeneic resistance plays an important role in natural resistance to this virus infection. Since the marrow-dependent effector cell appears to be closely related to the natural killer (NK) cells, an NK assay with HSV-1-infected fibroblasts [NK(HSV-1)] has been developed to study this resistance mechanism in humans. Incubation of effector and target cells for 12 to 14 h gave the greatest percent specific release (%SR) and kept spontaneous (51)Cr release from infected target cells below 35%. Patients with Bruton's agammaglobulinemia demonstrated significant kill indicating antiviral antibody was not necessary. Seropositive individuals gave a 9% greater%SR than seronegative individuals. Depletion of B-cells consistently diminished NK (HSV-1) for seropositive individuals and augmented kill for seronegative individuals. Although antiviral antibody produced in culture may contribute to NK (HSV-1), depletion of B-cells allowed quantitation of NK (HSV-1) to the exclusion of most of the antibody-dependent kill. The NK cells detected by this assay showed many of the properties reported for NK cells with K562 targets. Two patients with severe herpesvirus infections demonstrated NK (HSV-1) responses greater than 2 standard deviations below the normal mean. Since normal individuals with virus infections have higher rather than lower natural kill, the low NK (HSV-1) may reflect their susceptibility to the virus infection.     

Antiviral T-Cell-Independent Type 2 Antibody Responses Induced in Vivo in the Absence of T and NK Cells

Polyomavirus (PyV) infection induces protective T-cell-independent (TI) IgM and IgG responses in T-cell-deficient (TCR βxδ−/−) mice. In this study, we show that PyV is a TI -2 antigen: B cells with a mutated Bruton's tyrosine kinase (Xid mutants) do not respond to PyV with antibody secretion in the absence of T cells. We also demonstrate that NK-cell-mediated “help” is not absolutely required for the induction of the TI-2 antibodies to PyV; thus for the first time, we provide evidence for protective IgM and IgG responses against a viral infection induced in mice lacking T and NK cells (CD3Etg). Comparison of the antibody responses observed in T- and NK-cell-deficient mice with those of mice lacking only T cells, however, suggests that NK cells may promote isotype switching to IgG2a. This effect is probably mediated by IFNγ secretion. In support of this idea, studies on the antibody responses of PyV-infected SCID mice that had been reconstituted with IFNγR−/− B cells or wild-type B cells demonstrated the IFNγ dependence of PyV-specific TI IgG2a secretion and provided evidence that IFNγ acting directly on B cells plays an important role in TI pathways of isotype switching to IgG2a in vivo.     

Specific Inhibition of Antibody Production: II. Paralysis Induced in Adult Mice by Small Quantities of Protein Antigen

A state of immunological paralysis has been induced in adult CBA mice by intraperitoneal injections of small quantities of bovine γ globulin (BGG). The minimum paralysing dose of BGG has been found to be between 50 and 200 μg. A dose as small as 2 μg. has been found to have a slight paralysing effect. The time necessary for the induction of paralysis by 50 μg. to 2 mg. of BGG in CBA mice is 3–4 days.

Paralysis is induced by only one component of BGG; this component is incapable of inducing an antibody response unless an injection of adjuvant is made at the same time or slightly before the injection of the antigen. The BGG is centrifuged at an RCF of 20,000–30,000 g to remove particulate matter. Failure to remove the particulate matter leads to sporadic immune responses in groups of mice injected with the protein. Mice given a paralysing injection of BGG were subsequently challenged by an injection of BGG in Freund's adjuvant. The result of this challenge was tested by an injection of radioactively-labelled antigen and the elimination of this antigen from the circulation of the challenged mice was followed for several days. `Immune elimination' can easily be distinguished from `non-immune elimination'. The presence of antibody to the non-paralysing components of BGG in sera from paralysed mice was confirmed using the Ouchterlony geldiffusion technique.

     

Lack of Correlation between Mycoplasma Induced IFN-γ Production in vitro and Natural Killer Cell Activity against FLD-3 Cells

Interferon (IFN) stimulates natural killer (NK) cell-mediated lysis of tumor cells. However, it is not clear whether IFN production is essential for NK cells to lyse their target cells in vitro, especially in long-term (> 18 hrs) assays. To investigate this, 0.5 x 106 normal mouse spleen cells were cocultured in RPMI 1640 medium with Friend erythroleukemia cells (FLD-3) (1 x 104) for 24 hours under conditions which cause lysis of FLD-3 cells. Supernatant fluid from such cultures demonstrated antiviral activity (100-200 units) which could be identified as IFNy. Prior filtration of spleen cells over nylon wool, and pretreatment with anti-Thy-1.2 + C' abrogated their ability to generate IFN-y without affecting their NK (FLD-3) activity. The IFN-y producing cell which could also be detected in spleens of nu/nu BALB/c mice lacked cell surface, Lyt-1, Lyt-2, and NK-1.2 antigens. The stimulus for IFN-y induction appeared to be Mycoplasma arginini carried in the FLD-3 tumor cells. Although mycoplasma-free FLD-3 cells failed to induce IFN in vitro, they retained their susceptibility to NK cell-mediated lysis. We conclude that IFN induction is not essential for NK(FLD-3) cell-mediated lysis; indeed IFN detected in NK cell assays may be produced in response to mycoplasma infection of the tumor cells. The Thy-1.2 positive cells stimulated by mycoplasma to produce IFN-γ lack several characteristics of T-cells or NK cells.     

Correlation between TSH Receptor Antibody Assays and Clinical Manifestations of Graves' Orbitopathy

Purpose

To investigate an association between the levels of serum thyroid-stimulating hormone (TSH)-receptor autoantibodies (TRAbs) and Graves'' orbitopathy (GO) activity/severity scores, and compare the performance of three different TRAb assays in assessing the clinical manifestations of GO.

Materials and Methods

Cross-sectional study. Medical records of 155 patients diagnosed with GO between January 2008 and December 2010 were reviewed. GO activity was assessed by clinical activity score (CAS) and severity graded with the modified NOSPECS score by a single observer. Serum TRAb was measured by three different methods: 1^st generation thyrotropin-binding inhibitor immunoglobulin (TBII) assay (TRAb1^st); 3^rd generation TBII assay (TRAb3^rd); and biological quantitative assay of thyroid-stimulating immunoglobulin (TSI) using Mc4-CHO cells (Mc4-CHO TSI assay). Results were correlated with scores of activity/severity of thyroid eye disease.

Results

All three assays (TRAb1^st, TRAb3^rd, and Mc4-CHO TSI) yielded results that were significantly positively correlated with CAS (β=0.21, 0.21, and 0.46, respectively; p<0.05) and proptosis (β=0.38, 0.34, and 0.33, respectively; p<0.05). Mc4-CHO TSI bioassay results were significantly positively correlated with all GO severity indices (soft tissue involvement, proptosis, extraocular muscle involvement, and total eye score; β=0.31, 0.33, 0.25, and 0.39, respectively; p<0.05).

Conclusion

Mc4-CHO TSI bioassay was superior over the two TBIIs in assessing active inflammation and muscle restriction due to GO, whereas TBII assay would be sufficient for evaluation of patients with proptosis.     

Antibody Responses to Antigenic Sites 1 and 3 of Serotype 3 Poliovirus after Vaccination with Oral Live Attenuated or Inactivated Poliovirus Vaccine and after Natural Exposure

Three important antigenic sites involved in virus neutralization on polioviruses in mouse experiments have been identified. These sites are located at the surface of the virion and have been designated antigenic sites 1, 2, and 3. In mice, the antibody response to antigenic site 1 of serotype 3 poliovirus is considered to be immunodominant, but little is known about the immunogenicity of these sites in humans. In the present study, we developed inhibition enzyme-linked immunosorbent assays specific for antigenic sites 1 and 3 to measure antibody responses to these sites in fully vaccinated inactivated poliovirus vaccine (IPV) (n = 63) and oral live attenuated poliovirus vaccine (OPV) (n = 63) recipients and in naturally infected persons (n = 25). Similar levels of antibodies to site 1 in IPV and OPV vaccinees were detected. However, significantly more OPV recipients (88.7%) had detectable antibodies to antigenic site 3 (P < 0.01) than did IPV-vaccinated persons (63.1%). After an IPV booster vaccination, both previously IPV- and OPV-vaccinated persons responded with a significant increase in antibodies to sites 1 and 3 (P < 0.01). We conclude that the immune response to serotype 3 poliovirus in humans consists of both site 1- and site 3-specific antibodies and that these responses can be induced by either OPV or recent IPV vaccination.