Genetic studies of four highly homologous rheumatoid factor-associated Vk genes in rheumatoid arthritis patients and normal individuals.

Rheumatoid factors (RFs) are autoantibodies directed against IgG molecules. They are present in increased quantity in most patients with rheumatoid arthritis (RA), and are implicated in tissue damage in this disease. Paradoxically, recent studies of RFs have revealed that these autoantibodies are likely a physiological component of the immune system, and may play a role in the development and function of the B cell repertoire. Previously, we found that a significant fraction of RA patients express RF bearing the 6B6.6 cross-reactive idiotype, which is a phenotypic marker of the Humkv328-like genes. In order to elucidate the possible genetic factors that may contribute to the abnormal production of RFs in RA patients, we studied restriction fragment length polymorphisms (RFLP) of four highly homologous RF-related kappa light chain variable region (Vk) genes (i.e. Humkv328, Humkv328h2, Humkv328h5 and Humkv329) in RA patients and normal controls. The results show that kv328, kv328h2 and kv329 are likely to be alleles of the kv328 locus, while kv328h5 is a highly homologous Vk gene residing in a separate locus; and that deletion in one copy of either the kv328 or the kv328h5 loci, but not both loci, occurs in several individuals. However, the frequencies of various RFLP patterns of these two Vk gene loci are similar in patients and normals.

Reduced immunoreactivity to calcium-binding proteins in Purkinje cells precedes onset of ataxia in spinocerebellar ataxia-1 transgenic mice.

Earlier we have shown alterations in immunoreactivity (IR) to the calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CaB) in surviving Purkinje cells of patients with spinocerebellar ataxia-1 (SCA-1). In the present study we determined PV and CaB expression (by immunohistochemical and immunoblot analyses) in Purkinje cells of transgenic mice (TM) expressing the human SCA-1 gene with an expanded (line B05) and normal (line A02) CAG tract, as well as in age-matched nontransgenic mice (nTM). Heterozygotes in the B05 line develop progressive ataxia beginning around 12 weeks of age. A02 animals are phenotypically indistinguishable from wild-type (nontransgenic) animals. In the cerebella of 8-, 9-, and 12-week-old TM-B05 there was a progressive decrease in PV IR in Purkinje cells compared with nTM and TM-A02. Parvalbumin immunostaining in interneurons was well preserved in all groups. A progressive decrease was also observed in CaB IR in Purkinje cells of 8-, 9-, and 12-week-old TM-B05. Cerebellar Purkinje cells of 6-week-old TM-B05, which exhibit no ataxia and even lack demonstrable Purkinje cell loss, also revealed reduction in PV IR. This change was matched by a significant decrease in the amount of cerebellar PV in 6-week-old TM-B05 as determined by Western blot analysis. Calbindin D-28K immunohistochemistry did not detect any marked changes in CaB IR within Purkinje cells at 4 weeks. However, at 6 weeks immunostaining and immunoblot analysis revealed a significant decrease in CaB in TM-B05 compared with controls. These data suggest that decreased levels of calcium-binding proteins in Purkinje cells in SCA-1 transgenic mice may cause alteration in Ca2+ homeostasis.

Molecular genetic studies of rheumatoid arthritis.

Bam HI DR-beta and DQ-beta restriction fragment length polymorphisms (RFLPs) were found with increased frequency in white persons with seropositive rheumatoid arthritis as compared with control subjects. DR-beta 4.8-, 5.2-, and 7.0-kilobase (kb) RFLPs were observed in 86.5 percent of rheumatoid arthritis patients and in 56 percent of control subjects (p = 0.001, relative risk [RR] = 5.0). The 6.0-kb RFLP was present in 79 percent of rheumatoid arthritis patients and 32 percent of control subjects (p = 0.0002, RR = 8.0). The 4.8-, 5.2-, and 7.0-kb RFLPs correlated with DR4, -7, -9, and -w53 phenotypes and the 6.0-kb RFLP correlated only with DR4. Thus, these RFLPs do not appear to be disease-specific. A DQ-beta 3.2-kb RFLP was found in 63.5 percent of rheumatoid arthritis patients and in 38.0 percent of control subjects (p = 0.01, RR = 2.8). This fragment was frequently found in persons expressing DR1 and DQw1 phenotypes. Probes consisting of the first exon of the DR-beta-I and DR-beta-IV genes, respectively, only hybridized with the 5.2- and 6.0-kb RFLPs. These data suggest that more than one gene within the major histocompatibility complex contributes to susceptibility to seropositive rheumatoid arthritis in white persons.

Restriction fragment length polymorphism analysis in ankylosing spondylitis.

We utilized the technique of restriction fragment length polymorphism (RFLP) analysis in order to examine class I major histocompatibility complex genes in 52 Alabama ankylosing spondylitis patients and 107 local control subjects. A 9.2-kilobase PvuII RFLP was identified using the class I-specific B7 cDNA probe pDP001 that was closely associated with ankylosing spondylitis, most specifically with peripheral joint (including shoulder and hip) involvement. This fragment is associated with human leukocyte antigen A3 and A9 alleles, and segregation analysis in 11 multiplex families showed the RFLP to frequently segregate independently of B27 haplotypes. Two more recent studies have not confirmed the association of the 9.2-kilobase PvuII RFLP with ankylosing spondylitis per se, believed to be due to clinical and possibly genetic differences between the patient groups studied. These data strongly suggest at least one other major histocompatibility complex class I gene to be operative in predisposition to or modification of ankylosing spondylitis.

Peripheral blood chimerism in renal allograft recipients transfused with donor bone marrow.

Experimental studies have shown that administration of antilymphocyte serum combined with donor bone marrow cells can induce tolerance to allograft tissue. We have initially reported application of these protocols in clinical studies of cadaveric renal allograft recipients who were treated with MALG and donor-specific bone marrow cells. To evaluate the effectiveness of the donor marrow cells in the production of chimerism, a detection method based on 32P-incorporated PCR was established. The 32P PCR was utilized with primers specific for the HLA class II, VNTR (D17S5 and D1S111), and/or Y-chromosome genes to detect the presence of allogeneic chimerism in the recipients. Immediately posttransplant, 26.4% of marrow recipients demonstrated the presence of allogeneic chimerism prior to the marrow transfusion as did 18% in the untransfused controls. In transfused patients, chimerism was detected most frequently during the 1-3-month interval after marrow transfusion (65%), and then diminished to 50-56% at 3-12 months posttransfusion. In the control group the frequency of allogeneic chimerism was gradually decreased and was undetectable in the majority of the patients beyond 3 months posttransplant while marrow-transfused recipients were more likely to have chimeric cells detected consistently beyond 3 months. Rejection episodes were significantly effected by the presence of chimerism in the recipients. Of the transfused patients, 91.3% who demonstrated allogeneic chimerism were rejection-free as compared with 8.7% who experienced at least one rejection episode (P = 0.01). While the presence of allogeneic chimerism in the control group was correlated with rejection-free graft survival, this difference did not reach statistical significance.

Limiting detection of an amplification signal for HLA-D region and VNTR genes by 32P-PCR.

The limiting detection signal for identification of human genetic markers, such as HLA-D and VNTR genes, was determined using DNA isolated from a series of decreasing numbers of lymphocytes carrying the target marker in the polymerase chain reaction (PCR). The PCR procedure was assembled by incorporating 32P-labeled dCTP in the reaction mixture. Primers specific for detection of MHC Class II genes such as HLA-DR1, -DR2, -DRw52 and -DRw53 were utilized when cells were mismatched by one DR type, and primers for the identification of the region of variable number of tandem repeats (VNTRs) were utilized where cells had the same DR types. The 32P-incorporated amplified DNA was analyzed by polyacrylamide gel electrophoresis followed by exposure to x-ray film. The sensitivity of the test varied for different allelic markers as evaluated by amplification of DNA from each set of a mixture of lymphocytes. The target HLA-DR markers were detectable in a cell ratio of as high as 1:100,000, whereas the VNTR markers were detectable at a 1:1000 cell ratio. The approach described here offers certain advantages: 1) increased sensitivity, 2) quantitative power, 3) reduced assay time, 4) simplified procedure and 5) less expense. This method provides valuable information for studies involving forensic specimens and marrow engraftment after allogenic bone marrow transplantation (BMT) that require discrete representation of one allele relative to another in a heterozygous sample where limited quantities of target DNA are available.

HLA in ankylosing spondylitis: is HLA-B27 the only MHC gene involved in disease pathogenesis?

Linkage analysis of HLA haplotypes, HLA-B27 subtypes, and a 9.2-kb PvuII B7 restriction fragment length polymorphism (RFLP) (shown previously to be associated with peripheral arthritis in ankylosing spondylitis [AS]) in 115 relatives from 12 multiplex spondyloarthropathy (SNSA) and 2 B27-positive control families showed AS to be linked to HLA-B27 haplotypes. The RFLP segregated with HLA-A3- and HLA-A9-bearing haplotypes (lod score, 10.98; odds in favor of linkage, 9.2 x 10(10):1), although its linkage to AS or other SNSA per se was not seen. The association of HLA-A3/A9 with the RFLP was also seen in 52 AS patients and 92 controls, although no HLA-A, -B, -C, or -DR allele (other than HLA-B27) was significantly increased in frequency. The HLA-B27 subtype seen on all but one of the haplotypes studied was B*2705, the sole exception being HLA-B*2702. Although not ruling out a second HLA-A-linked gene influencing the clinical expression of AS, these data fail to support the role of a second MHC-associated gene in the pathogenesis of AS per se.

Analysis of restriction fragment length polymorphisms in rheumatic diseases.

Considerable evidence indicates that genes residing within the major histocompatibility complex (MHC) influence susceptibility to certain rheumatic diseases, such as ankylosing spondylitis (AS) and rheumatoid arthritis (RA). However, it has not yet been possible to precisely identify the gene(s) responsible for conferring enhanced susceptibility to these diseases. The availability of recombinant DNA technology should accelerate progress in obtaining this goal. A particularly promising method in this regard is restriction fragment length polymorphism (RFLP) analysis using appropriate class I and class II MHC gene probes. In preliminary studies, RFLPs have been identified for AS and RA which associate with susceptibility to the disease. Further studies using this approach should permit localization and precise identification of the disease susceptibility gene(s) for these diseases.

Donor specific bone marrow cells suppress lymphocyte reactivity to donor antigens and differentially modulate TH1 and TH2 cytokine gene expression in the responder cell population.

Previous studies have shown that post-transplantation infusion of donor specific bone marrow following a non-specific potent immunosuppressive agent such as antilymphocyte globulin (ALG) can significantly enhance graft survival compared to ALG alone. This enhancement remains variable and is thought to occur through the induction of specific partial tolerance to the renal allograft, but the underlying cellular mechanisms have not been clearly identified. In order to improve the efficacy of this specific immunosuppressive treatment and to study the events leading to enhanced allograft survival, we sought to establish a simple in vitro model based on a mixed lymphocyte reaction (MLR). We show that cellular proliferation seen in a normal MLR can be suppressed by addition of donor specific bone marrow cells (BMC). Significantly, this suppression is not observed with either third party BMC or donor specific peripheral blood mononuclear cells (PBMC). We have defined the optimum conditions of bone marrow infusion regarding number of BMC, their handling and culture, and simple enrichment procedures. Using a semiquantitative polymerase chain reaction assay, we have studied the cytokine gene expression in MLR modulated by donor specific BMC. In an unmodified allogeneic response, the responder cells show increased expression of interleukin-2 (IL-2) gamma-interferon IFN-gamma and receptor (IL-2R) mRNA, and no IL-10 mRNA. When responder cells are cultured with BMC of the stimulator, there is a 256-fold decrease in IL-2 mRNA, and a 64-fold decrease in IFN-gamma and IL-2R mRNA. There is also a 64-fold increase in IL-10 mRNA. This effect is even more marked when the BMC are depleted of CD3+ cells. The kinetics of addition of donor specific BMC to the normal allogeneic MLR culture and specificity of the action of BMC are also elucidated. Our data suggest that the enhancement of graft survival observed with donor BMC may operate through decreased proliferation of reactive T cell clones (due to decreased IL-2/IL-2R) and suppressed monocyte functions (due to decreased IFN-gamma and increased IL-10 gene expression).

Combined analysis of cytokine genotype polymorphism and the level of expression with allograft function in African-American renal transplant patients.

Cytokine gene polymorphism and expression levels were evaluated in a group of African-American patients who had undergone renal transplantation. It was hypothesized that possession of specific cytokine alleles might be influential in predisposing the recipient to allograft rejection. Thus, we sought to establish a relationship between cytokine gene polymorphism, the levels of cytokine expression, and the outcome of allograft function. Cytokine genotypes and mRNA transcript levels of IL-2, TNF-alpha, TGF-beta1, IL-10, IL-6 and IFN-gamma were determined using peripheral blood cells. Genomic DNA samples from 77 transplant recipients and 77 controls were tested by a multiplex PCR with specific primers for the above cytokines. The frequency distributions of cytokines were analyzed in respect to the clinical characterization, including delayed graft function (DGF), rejection episodes (REs) and stable graft function (SGF). The mRNA transcript level was tested both at pre- and early post-transplantation (day 1 and day 4) with primers for coding regions of the above cytokines in a RT-PCR assay. The majority of recipients with successful graft function were matched with their donors for only three out of the six HLA alleles. We have shown that the TGF-beta1 T/C G/G high producer and IFN-gamma T/A intermediate producer genotypes were associated with allograft rejection, whereas low IFN-gamma producer and high IL-10 producer genotypes were significantly protective of the allograft. There was some correlation between the TGF-beta1 high producer genotype and DGF, but it was not statistically significant. Overall, 77% of those who experienced REs carried the TGF-beta1 T/C G/G, high producer genotype as compared with 52% who experienced DGF, 39% with SGF (P<0.01, RR=2.0), and 27.3% of controls (P<0.003, RR=2.6). The IFN-gamma T/A intermediate producer genotype was found in 69.2% of patients with REs as compared with 26.8% of patients with SGF (P<0.008, RR=2.85). The IL-10, ATA/ATA low producer genotype was found in 38.5% of recipients with REs and 14.6% of recipients without REs (P<0.04, RR=0.53). Expression levels of mRNA transcript were correlated with genotype data, except for the TGF-beta1 high producer genotype where there was no significant difference between the level of mRNA transcript at pre- and post-transplantation. Low DRbeta1 and high DPbeta1 expression by recipient peripheral blood mononuclear cells before transplantation was associated with more SGF, whereas high DRbeta1 and low DPbeta1 expression at pretransplantation was associated with more REs (DRbeta1, P<0.001 and DPbeta1, P<0.05, respectively). We concluded that, dual analysis of cytokine genotype and expression levels by peripheral cells may be an important clue to understanding the contribution of the recipient's immune response to an allograft pre- and post-transplantation. Identification of peripheral markers diagnostic of rejection could allow advance anticipation of clinical outcome, and might reduce the need for tissue biopsy.

Relationship between ataxin-1 nuclear inclusions and Purkinje cell specific proteins in SCA-1 transgenic mice.

Spinocerebellar ataxia-1 (SCA-1), like other polyglutamine diseases, is associated with aggregation of mutant protein ataxin-1 in the nuclei of susceptible neurons. The role of ataxin-1 aggregates in the pathogenesis of susceptible neurons, especially cerebellar Purkinje cells, is unknown. The present study was initiated to determine the temporal relationship between ataxin-1 aggregation and the sequence of specific biochemical changes in Purkinje cells in SCA-1 transgenic mice (TM). Earlier, we demonstrated that SCA-1 TM with no Purkinje cell loss and no alterations in home cage behavior show decreased expression of calcium-binding proteins calbindin-D28k (CaB) and parvalbumin (PV) in Purkinje cells. To determine if increased expression of mutant ataxin-1 in TM is also associated with earlier biochemical changes in Purkinje cells, both heterozygous and homozygous (B05 line of SCA-1) TM were used. The age of onset of ataxia in SCA-1 TM was at 12 weeks in heterozygotes and 6 weeks in homozygotes. In 6 week old heterozygous TM, Western blot analysis of growth associated protein 43 (GAP-43) and synaptophysin revealed no significant alterations as compared with the age-matched nontransgenic mice (nTM), whereas CaB was significantly reduced. beta-III-Tubulin was used as a specific Purkinje cell marker protein, immunohistochemical localization showed strong beta-III-tubulin immunoreactivity (IR) in Purkinje cells in 6 week old heterozygous TM, whereas CaB and PV IR were markedly reduced in the same neurons (double immunofluorescence staining). Most Purkinje cells from heterozygous (12 weeks old) and homozygous (6 weeks old) TM contained ataxin-1 nuclear inclusions (NIs). Cells with and without visible NIs revealed reduced PV and CaB IR; however, the changes were overtly more severe in cells with visible NIs. In contrast, the same cells were strongly immunoreactive to beta-III-tubulin. CaB, which is also present in the nucleus, colocalized with ataxin-1 and ubiquitin positive NIs. Further, RT-PCR analysis of CaB mRNA in the cerebellum in 6 week old heterozygous TM demonstrated a significant decrease in mRNA in comparison with the aged-matched nTM. These data suggest that there are selective alterations in the expression of CaB and PV in Purkinje cells which possibly occur earlier than ataxin-1 aggregation. Further, we speculate that ataxin-1 aggregates may not be toxic in general; however, they may deplete specific proteins essential for Purkinje cell viability in SCA-1 TM.

Calcium homeostasis and spinocerebellar ataxia-1 (SCA-1).

Spinocerebellar ataxia-1 (SCA-1) belongs to a group of polyglutamine neurodegenerative disorders characterized by the expansion of a glutamine tract within the mutant disease-causing protein. In SCA-1, the expression of mutant ataxin-1 induces a progressive functional loss and the subsequent degeneration of a set of neurons including cerebellar Purkinje cells. Studies on SCA-1 transgenic mice have provided further understanding of the molecular and cellular events important for the disease. This review discusses what has been learned about the pathogenesis of SCA-1 through the transgenic mouse models in reference to Ca(2+) dependent pathways. This article also discusses the role of Ca(2+) regulating cytoplasmic and nuclear proteins in the pathogenesis of SCA-1. Finally, we discuss the use of double mutant mouse models to understand the association between Ca(2+) binding proteins and Purkinje cell pathology in SCA-1.

Polymerase chain reaction for the detection of bacteremia.

Analysis of blood by polymerase chain reaction (PCR) is a more rapid and sensitive method to detect bacteremia than blood culture. The PCR was performed on blood obtained from patients during blood culture draws and on blood from normal volunteers. Eighty-seven patients provided 125 blood samples for blood culture comparison with PCR. Specific PCR primers for Staphylococcus aureus and Escherichia coli that targeted conserved regions common to gram-positive and gram-negative bacteria were used. Selective stringency conditions identified other gram-positive and gram-negative bacteria. The blood culture agreed with the PCR in 93 of the 125 patient specimens (74%). In 29 of these specimens the PCR was positive yet the blood culture was negative. When clinical information was included with positive blood culture to define sepsis in these patients and their specimens were added to the positive blood cultures the statistical accuracy of PCR was 93 per cent. Only three of the 78 specimens with negative PCR had positive blood cultures. The PCR was negative in all but one of the 50 volunteers. PCR is more sensitive than blood culture, and it can quickly rule out bacteremia.

Early expression profile of inflammatory markers and kidney allograft status.

Cellular rejection after renal transplantation, in general, occurs as a result of an interaction between immunologic processes that maintain graft tolerance versus allograft rejection. A potential mechanism that triggers such processes might be through the activation of the innate immune response initiated during organ procurement and ischemia/reperfusion injury, contributing to delayed graft function or graft dysfunction. Our goal was to test the impact of molecular markers that have key roles in innate immunity such as cytokines, Toll-like receptors (TLRs), and allograft inflammatory factor-1 (AIF- 1) at early times after transplantation. Blood samples from a total of 90 patients who received kidney transplants were included in this study. Three samples from each patient at different time intervals (pretransplantation, day 3, and day 6 after transplantation) were tested using a quantitative reverse transcriptase polymerase chain reaction. The mRNA transcripts were tested in association with glomerular filtration rates (GFR) as a measure of allograft function. Surgical samples obtained from transplant nephrectomy were used in a tissue array for immunohistochemistry testing. In peripheral blood mononuclear cells, the mean ± standard error of mean (SEM) for interleukin 18 (IL-18), and IL-10 mRNA expression were increased and interferon-γ was decreased in association with high GFR post-transplantation as compared with the pretransplantation expression levels. The mean ± SEM for expression level of AIF-1 was increased 1.5-fold and for TLR-2 and TLR-4 were increased 1.2 to 1.4-fold in samples obtained on day 6 post-transplantation in association with low GFR (P < .05). In neutrophils, the mean ± SEM levels of TLR-2 mRNA was increased 2-fold on day 6 in association with high GFR (P < .005), but was reduced 2.8-fold in association with low GFR (P < .002). In conclusion, the mRNA profiles of biomarkers presented here appeared to be informative for prediction of allograft status and outcome.

Cardiac allograft rejection correlates with increased expressions of Toll-like receptors 2 and 4 and allograft inflammatory factor 1.

BACKGROUND: Evidence suggests that injury-induced activation of the recipient's innate immune response determines the outcome of allograft transplantation. The mechanism responsible for the induction of such innate immune response is not clear yet. We hypothesized that in cardiac transplantation settings, the initial myocardial ischemia and postischemia graft reperfusion may release allograft inflammatory factor (AIF) 1, causing Toll-like receptor (TLR)-mediated activation of macrophages and dendritic cells, leading to the production of cytokines and the activation of adaptive alloimmunity. Therefore, our goal was to validate the presence of these biomarkers in the peripheral blood and biopsy specimens of patients presenting allograft rejection. METHODS: We studied 90 peripheral blood and 30 endomyocardial biopsy specimens from patients who had undergone cardiac transplantation. Specimens were tested by quantitative reverse-transcription polymerase chain reaction to determine TLR-2 and -4 and AIF-1 expression levels, correlating with clinical rejection grades. The group differences for mRNA transcript levels between the rejection grades were determined by 1-way analysis of variance. The level of significance was set at P < .05 for comparison between the groups. RESULTS: The mean ± SEM level of TLR-2 mRNA expression was increased 1.7-fold in monocytes (P < .05) and 4.2-fold in biopsy samples from groups with grade 3A compared with grade 1A or grade 0 rejection (P < .0001). AIF-1 expression was increased 2.4-fold in monocytes (P < .05) and 4.2-fold in biopsy samples comparing grade 3A versus 1A rejections. The TLR-4 mRNA expression was also increased in the group with 3A rejections; however, the difference was only significant in biopsy specimens (P < .0001). CONCLUSIONS: Our data demonstrated that expression profiles of AIF-1 and TLR-2 correlated with biopsy-proven allograft rejection in both peripheral blood and local tissue, suggesting their potential as diagnostic biomarkers for early detection of allograft rejection.

A new HLA-DPA1 allele, DPA1*02016, identified in African-American population.

A new HLA-DPA1 allele has been found in the African-American population. This newly discovered type is homologous with the DPA1*02011 allele, except in codon 38 of the DPA1 sequence. The variant of DPA1 was detected by sequence-specific oligonucleotide (SSO) typing, and specified by cloning and sequencing methods. GenBank accession number is AF165160. The WHO Nomenclature Committee has officially assigned the name DPA1*02016.

Dominantly inherited ataxias.

Molecular genetic studies in the past decade have demonstrated the enormous genetic heterogeneity among the dominantly inherited ataxias. Mutations at several distinct loci give rise to the progressive dominant ataxias and at least 2 different mutations cause episodic ataxias with dominant inheritance. The well-established genotypes for progressive dominant ataxias have all involved expansions of repeated CAG sequences. Clinically these patients present with progressive cerebellar deficits as well as signs relating to pathology in other neural systems in a variable fashion. Some of these other signs serve as diagnostic clues to the underlying genotype, but the identification of the genotype from the clinical phenotype alone is usually difficult. The CAG expansions involved usually are unstable with intergenerational expansions as well as contractions of the repeat size. Phenotypic features such as age of onset and to a lesser extent disease progression rate and the presence of specific clinical signs depend on the CAG repeat size. Identification of the mutations has allowed precise genotypic diagnosis in several families allowing more accurate genetic counseling, including predictive testing of at risk individuals when sought. Also, increasing information about the gene products and their abnormal distributions in disease brain is rapidly giving rise to rational ideas about the pathogenesis of neuronal degeneration in these diseases and raising hope for meaningful treatment strategies.

Localization of protection-eliciting epitopes on PspA of Streptococcus pneumoniae between amino acid residues 192 and 260.

Pneumococcal surface protein A (PspA) is a virulence factor of Streptococcus pneumoniae that can elicit a protective antibody response. The pspA gene of strain Rx1 encodes a 65 kDa molecule composed of 588 amino acids. The N-terminal 288 amino acids are highly charged, and predict an alpha-helical coiled-coil protein structure. All monoclonal antibodies (MAbs) to PspA, obtained by screening against whole pneumococci, bind to the alpha-helical region of PspA, suggesting that this region is surface exposed. The C-terminal 217 amino acids of PspA contain the surface anchor of PspA and does not appear to be alpha-helical. In the middle of the molecule is a proline-rich region that is thought to traverse the cell wall. In this study we have mapped the immunogenic epitopes detected by 9 MAbs that were made against strain Rx1 PspA. Five of the MAb also react with the PspA of mouse virulent strain WU2. All epitopes were found in one of two portions of the alpha-helical region. One comprised the first 115 amino acids, and the other was within amino acids 192 and 260. The five MAbs that recognize WU2 PspA, but not the remaining four MAbs, were protective against strain WU2. The epitopes detected by four of the five protective MAbs mapped to region 192 to 260 of Rx1 PspA. The existence of protective epitopes in this region was confirmed by demonstrating that mice immunized with the cloned fragment containing these residues were protected from fatal infection with WU2. Since amino acids 192 to 260 are in the region of PspA anticipated to be adjacent to the cell wall, and probably well covered by capsule, the means by which antibodies to the region lead to protection is not obvious.