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The HLA-A, -B and -C locus gene frequencies and the significant A, B and B, C haplotypes of the population of Campania (Southern Italy) are reported. From a comparison of gene frequencies and from an overall genetic distance evaluation with other European and Mediterranean populations the typical Mediterranean structure of the Campanian population is confirmed. While keeping several Middle Eastern features, it places itself as far from Northern Italy (Bergamo) as from the Spanish and the French populations. Among all the groups compared, including Turkish and Lebanese, the largest distance is measured from the English, the Danish and the German populations.

Human peripheral T cells were fractionated in accordance to their surface receptors for G or M immunoglobulins and analyzed for their reactivity with alloantisera obtained by planned immunization involving HLA-A and HLA-B compatible individuals. These alloantisera were previously shown to recognized polymorphic structure exclusively expressed on T cells. Although the majority of the alloantisera analyzed reacted with different proportions of both T(M) and T(G) populations, some antisera specifically recognized surface structure restricted to either T(M) or T(G) lymphocytes. The various alloantisera consistently reacted with a fraction only of T(G) and T(M) cells, this indicating that these T cell subsets can be further fractionated in accordance to the expression of the alloantigenic determinants recognized by these antisera.

Using two alloantisera produced by planned immunization of blood donor volunteers, a new specificity was defined residing on human Ia molecules. The specificity is possessed by the following human lymphoid cell lines: Reh, NALM-1, B89, KM-3 and BJAB, and appears to be the same as the DRw13 specificity discovered in the 8th International Histocompatibility Workshop. A radioimmunoassay for typing w13 on peripheral blood lymphocytes was established. A population and family study supports the genetic control by the HLA-DR locus. The phenotypic frequency in an Italian population was 3.7 percent. The relationship between w13 and the MT2 supertypic specificity was confirmed at the molecular level and the papain sensitivity of w13-carrying molecules was determined.

Sixty patients with celiac disease were typed by radioimmunoassay for the specificities HLA-DR3, HLA-DR7, and for an allelic specificity, DC3, of the HLA-associated DC locus. We found that celiac disease is primarily associated with the DC determinant. The previously described associations with DR3, DR7, B8, B13, and A1 can be explained by decreasing degrees of linkage disequilibrium with DC3.

Analysis by the indirect fluorescence test followed by fluorescence-activated cell sorter (FACS) analysis has shown that antisera recognizing subsets of human T lymphocytes can be produced by planned immunizations involving HLA-A and HLA-B compatible donors. The reactivity of these antisera against some individuals of a population but not others shows that they recognize a polymorphic cell surface component. The reactive subpopulation largely overlaps with the JRA+ subset, which was previously shown to possess regulatory properties in functional assays. The specificity of the antisera for a T cell subset and the unrelatedness of the anti-B cell activity of the same antisera has been confirmed by two-color fluorescence tests.

Antisera recognizing subsets of human T lymphocytes can be produced by planned immunizations involving HLA-A and HLA-B compatible donors. The reactivity of these antisera against some individuals of a population, but not others, show that they recognize a polymorphic cell surface component. The study of informative families shows the HLA linkage of at least some of these alloantigenic determinants.

A DP serological allospecificity was identified using 125I-labeled preparations of HLA class II molecules isolated from cells of HLA homozygous typing cell lines, SLE (DRw6, DQw1, DPw3) and WT46 (DRw6, DQw1, DPw2), and depleted of DR molecules by absorption with an anti-DR monoclonal antibody. The specificity, provisionally called WT, was carried by class II molecules possessing the characteristics of DP molecules on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected primarily in DPw1, DPw3 and DPw5 cells and exceptionally in some DPw2 cells including WT46 and DPw4 cells on a large panel of DP-pretyped B cell lines mostly derived from the 10th International Histocompatibility Workshop B cell reference panel. It was apparently allelic to another DP serological specificity BUT previously defined on DP molecules isolated from cells of DPw2+ HLA deletion mutant cell line LCL 721.82. On the same cell panel; the BUT specificity was negative in all DPw1, DPw3 and DPw5 cells, and positive in all DPw2 and DPw4 cells and also in DPw2B and DPw4B cells except the cells typed WT+. This DP association pattern was similar to that of the known allelic sequences, GGPM and DEAV, in DP beta F segment, one of the six variable segments in the second exon of DP beta gene. Thus, genomic DNA from 22 B cell lines pretyped for BUT and WT specificities were enzymatically amplified for the second exon of DP beta gene by the use of locus-specific oligonucleotide primers and hybridized with 32P-labeled oligonucleotide probes corresponding to the F segment sequence variations. This oligonucleotide typing showed perfect one-to-one correlation with the BUT and WT serological typing. The typing also revealed that WT46 cells, although typed DPw2, have the DEAV sequence common to DPw1, DPw3 and DPw5.

IgM molecules were purified by the use of anti-IgM antibody-coupled Sepharose from the culture supernatant of an Epstein-Barr-virus-transformed lymphoblastoid cell line, MP1, that secretes alloantibodies possessing HLA-DQw2 specificity as defined by the cytotoxicity assay. The obtained IgM preparation was labeled with radioactive iodine-125I and fractionated by gel filtration. It contained pentameric IgM and smaller oligomeric IgMs. When tested by the direct cellular binding assay against a panel of HLA-typed cell lines, they all showed the DR3 and DR7 association pattern characteristic of DQw2. A weak but significant binding was detected for DR1, DR6, and DR9. On isoelectrofocusing, MP1 pentameric IgM gave a restricted banding pattern comparable to monoclonal IgM obtained from a patient with Waldenström's syndrome. Moreover, the pattern was identical to that of IgM purified from the culture supernatant of a defined hybrid clone, 162, that was generated by fusing MP1 cells with heteromyeloma D33 cells. The target class II molecules showed the dimeric structure that conforms to DQw2 molecules.

T cells recognize protein antigens as fragments (peptides) held in a defined binding site of class I or class II major histocompatibility (MHC) molecules. The formation of complexes between various immunologically active peptides and different MHC molecules has been demonstrated directly in binding studies between the peptides and solubilized, purified molecules of class II MHC. Studies with intact cells, living or fixed, have not directly demonstrated the binding of the peptides to MHC molecules on antigen-presenting cells, but the formation of such complexes has been shown indirectly through the capacity of antigen-presenting cells to stimulate specific T cells. Here we report evidence that supports directly the binding of radiolabelled influenza matrix peptide 17-29 to products of the human class II MHC locus HLA-DR, on living homozygous B-cell lines, and we show that the kinetics of such binding is much faster with living cells than with fixed cells. Furthermore, whereas the peptide reacts with HLA-DR molecules of all alleles, it binds preferentially to DR1, the restricting element in antigen presentation.

The la subset that reacts with alloantiserum HON known to possess a strong anti-DRw53 activity was isolated from a 125I-labeled Ia preparation obtained from cells of RPMI 8057 cell line (DR1,4) and was found on peptide mapping to be lacking in the pattern characteristic of DR-like molecules carrying the DRw53 specificity and to display the structural features of DQ molecules, particularly those carrying the DQw3 specificity. Distribution analysis on a panel of selected la-positive cell lines indicated that the specificity involved is associated only with DR4 and DRw9, differing from the known DRw53 pattern (DR4, 7, and w9) and also from the known DQw3 pattern (DR4 and 5). Reciprocal sequential binding experiments demonstrated that the HON-defined specificity resides along with DQw3 specificity on the same molecules. Thus, HON alloantiserum possesses two different antibody activities; one directed to DRw53 specificity and another directed to a new DR4- and w9-associated DQ specificity.

Two 8th Workshop alloantisera, 8W1062 and 8W614, which were classified respectively as anti-DR5 and anti-DR3, were found to react with an Ia preparation from LG38 cells (DR5,5) that was depleted of DR and BR molecules and enriched in DC molecules by pretreatment with a monoclonal antibody and an antiserum against alpha-subunits of DR and BR molecules. The reaction of both alloantisera was inhibited by DR3-positive, DR5-positive, and DRW13-positive individuals. This pattern does not correspond to any of the hitherto reported supertypic specificities. Both antisera were shown to bind to the alpha-subunit isolated from the DC-enriched preparation but not to the beta-subunit. The specificity has been named DC alpha 3. Family studies show the segregation according to the HLA haplotype patterns. This demonstrates by formal genetics criteria the HLA linkage of the locus controlling the DC alpha-subunit.

Human Ia molecules were isolated from cells of LG-38, an HLA-homozygous lymphoid cell line of DR5 specificity. Three Ia subsets could be distinguished and separated by using specific alloantisera and a monoclonal antibody with polymorphic reactivity. These subsets carried the specificities DR5 and MT2, MT2 alone and MB3 alone. The structure of the three molecular species was analyzed by microfingerprinting. The subset carrying only MT2 was similar, in both the component alpha and beta chains, to the major subset carrying DR5 and MT2, whereas the subset carrying MB3 was distinct in both chains from the other two subsets. These data are compatible with our previous findings obtained for the products of two Ia loci closely linked to the DR locus and provisionally called DC and BR; they also support the conclusion that the subset carrying only MT2 is an allelic product of the BR locus, whereas the MB3 subset is an allelic product of the DC locus. MT2 appears to be a shared specificity of DR and BR loci products.

Human peripheral blood lymphocytes respond in vitro to sheep red blood cells (SRBC) with the appearance of numerous specific plaque-forming cells, if the Epstein-Barr virus (EBV) is added to the cultures together with the antigen. However, it was found that when polyethylene glycol (mol. wt. 6000) is included in the culture medium at a final concentration of 4%, antigen alone is able to induce an anti-SRBC response, with clear hemolytic plaques of regular size. Addition of EBV causes a further increase in the antigen-specific antibody response.

Three distinct molecular subsets with different structures and alloantigenic determinants were identified in human Ia antigens from cells of an HLA-Dw7 homozygous cell line. The subsets carried DR7 specificity, BR4X7 supertypic specificity and MB2 supertypic specificity, respectively, and were immunospecifically separated by the use of operationally monospecific alloantisera. These specificities showed HLA-linked segregation in families and they were distributed in the population according to different but partially overlapping patterns. On peptide mapping analysis, the three subsets showed marked differences in the beta-chains. The alpha-chains of DR7 and BR4X7 subsets were very similar to each other, whereas the alpha-chains of MB2 subset were distinctive from those of DR7 and BR4X7. These data indicate the presence of a minimum of three HLA-linked loci; DR locus, a locus that encodes BR4X7, and a locus that encodes MB2, and substantiate the three-loci concept for the genetic control of human I antigens.

Human Ia(-like) specificities controlled by gene loci other than HLA-DR were searched for at the molecular level in cells of human B-cell-type cell lines which carry two established DR specificities. Chevalier cells of DRw3 and 7 and U698M cells of DRw2 and 4 were used. Their Ia molecules were partially purified, radioiodinated and analyzed for Ia specificities by the direct binding and sequential binding assays with a selected panel of human Ia alloantisera. It was possible in both the cell lines to define a third subset of Ia molecules carrying a new specificity in addition to two Ia subsets carrying the established DR specificities. The new specificity was detected by putative anti-DRw4 and anti-DRw7 antisera and was closely associated with DRw4 and DRw7 at population level. It was thus designated provisionally as BR4X7. These results suggest that the BR4X7 specificity is coded for by a separate Ia locus closely linked to HLA-DR locus. The determinant(s) responsible for BR4X7 was located on the small subunit of Ia molecules.

HLA-DP genotyping was performed using dot-blot analysis with synthetic oligonucleotide probes. Fourteen probes were designed based on the known sequence variations in the polymorphic segments of the DP beta second exon. Each probe was tested against genomic DNA amplified by the polymerase chain reaction, using DP beta-specific primers. A total of 45 HLA homozygous B-cell lines, selected from the Tenth International Histocompatability Workshop and pretyped for the known DP omega specificities, were analyzed. Different hybridization patterns were found for each DP omega specificity. The oligonucleotide hybridization performed on DP omega-negative B-cell lines gave a pattern distinct from those of known DP omega specificities, indicating the presence of novel DP allelic sequences. The use of sequence-specific oligonucleotides combined with DNA amplification allows a simple and reliable genotyping of DP antigens.

Three new alloantigenic specificities of human major histocompatibility complex class II molecules have been defined by testing the reactivity of alloantisera at the molecular level. Two of these specificities identify different DR4 haplotypes. The Fe75 specificity is associated with the DR4/Dw10 haplotype and the CBC/MRG6 specificity with the DR4/DKT2 haplotype. Both are supertypic specificities and are associated with other DR specificities as well. Both specificities are carried by class II molecules belonging to the first DR subset. Together with previously described determinants, these specificities contribute to serological discrimination of the different DR4 haplotypes.

A standardized procedure is proposed for deliberate immunizations against human major histocompatibility complex determinants. The data presented demonstrate its effectiveness and, by using a number of necessary precautions, this procedure has proven to be very safe. The following points are especially important: (1) exclusive utilization of regular blood donors as immunizers; (3) use of whole blood as an immunizing agent; and (3) use of small immunizing stimuli rather than large transfusions. This procedure can be recommended for the production of monospecific anti-HLA antisera and it may be useful if and when a deliberate transfusion policy for prospective kidney recipients is adopted.

Knowledge of the tumor content of estrogen (ER) and progesterone (PgR) receptors has proved to be of significant value in human breast cancer. Relative determinations were performed in 589 specimens in our laboratory. The positivity of ER and PgR is correlated with the patients' age at diagnosis, tumor size and relative grade. In particular, the significance of PgR versus ER status and the possible prognostic role of these receptors are investigated.

This study was carried out to assess the diagnostic accuracy of a new gastrointestinal cancer antigen ( GICA ) defined by a monoclonal antibody. Its sensitivity and specificity were assayed in a group of patients with different pancreatic diseases (10 acute pancreatitis, 27 chronic pancreatitis, 22 cancers of the pancreas) and in 29 normal individuals. The concentration of GICA was always inferior to 37 units/ml (our discriminant limit between cancer and noncancer patients) both in cases with chronic pancreatitis and in healthy subjects. Increased levels of the antigen were found in 16/22 (72.7%) pancreatic cancer patients and in 3/10 (33.3%) cases with acute pancreatitis. The assay was within the normal range in 2 (28.6%) out of 7 cancers judged resectable. The test is simple and rapid, but its relative sensitivity and the frequent elevation of GICA in other adenocarcinomas of the gastrointestinal tract make it unsuitable for screening programs in pancreatic cancer. Even its use for early diagnosis of cancer of the pancreas does not seem promising. The major finding of our study is the lack of false-positives in patients with chronic pancreatitis and therefore the usefulness of this test in differentiating preoperatively between chronic inflammation and cancer of the gland. Frequent increase of the marker in patients with acute pancreatitis is not yet clear.

Human class II molecules were isolated from cells of a DR2/Dw2-homozygous cell line, PGF. The three mutually exclusive subsets were separated by selective binding with monoclonal antibody MCS7 and alloantisera CCB921 and KY22. The specificity involved in the binding with alloantisera was identified to be a supertypic specificity associated with DR1, 2, and w9 for CCB921 and the DQw1 specificity known to be associated with DR1, 2, w6, and w10 for KY22. The MCS7 specificity appeared to be a cross-reactive specificity related to the DRw52-like specificity. On peptide mapping, the alpha chains of MCS7- and CCB921-reactive subsets were the same, showing the pattern characteristic of DR alpha chains, whereas the beta chains were very similar to, but distinguishable from, each other. These structural features conformed to those of DR or DR-like subsets. The KY22-reactive subset was distinctive in both alpha and beta chains from the above two subsets, and it displayed peptide patterns typical to DQw1-bearing Ia molecules. Interestingly, the MCS7- and CCB921-reactive subsets both carried the DR2 specificity, as indicated by their binding to alloantiserum Fe73/22 which was proven to be DR2-specific.

This paper describes the in vitro production of the human alloantibody 454A.C5 with HLA-DQw2 specificity produced by a stable EBV-transformed cell line. Immune B cells for transformation were generated by planned immunization of a blood donor volunteer with repeated HLA-immunizations over the course of 9 yr. Reactivity of 454A.C5 was in exact concordance with the presence of the DQw2 antigen as defined by microcytotoxicity assay on a panel of 21 B lymphoblastoid cell lines with well-characterized HLA antigens. Family studies showed the segregation according to the HLA-DQw2 haplotype; thus we demonstrate by serological and formal genetics criteria, that the alloantibody is specifically directed against the DQw2. 454A.C5 antibody may be employed as a monospecific HLA typing reagent.

Peripheral B lymphocytes obtained from three healthy individuals who had been immunized against peripheral blood lymphocytes from appropriate HLA-incompatible donors were transformed by the use of Epstein-Barr virus. The transformed blastoid B cells were repeatedly subcultured by means of "cluster picking," and the HLA antibody-producing cultures were identified by testing the culture supernatants by means of the cytotoxicity assay, using the corresponding donor cells. Thus far, four cell lines that secrete cytotoxic HLA antibodies (MP1, 3, 4, and 5) have been established. Specific immunoabsorption experiments revealed that the antibody activity is carried by lambda-type IgM for MP1, by kappa-type IgM for MP3 and MP5, and by both for MP4. Specificity analysis of a panel of HLA-pretyped cells indicated that MP1 detects DQw2, whereas MP5 recognizes B7. The specificity of MP3 was similar to a DQ specificity termed DC5 (probably equivalent to TA10) but not the same. In the case of MP4, both of the lambda-type and kappa-type antibodies appeared to be directed toward new HLA class II determinants.

Peripheral mononuclear cells (MNC) of patients with autoimmune thyroid disease have been shown to proliferate when cultured with human thyroglobulin (hTg). In addition, such a phenomenon is apparent in a certain number of healthy individuals. In this study we have attempted to correlate hTg-induced MNC proliferation, occurrence of anti-hTg autoantibodies and HLA phenotype (including Class II DR and DQ loci) in a population of HLA-typed normal blood donors. Fourteen out of 56 subjects showed a significant MNC proliferation to hTg. Three of them had anti-hTg autoantibodies in the serum, while none of the hTg-unresponsive subjects showed such antibodies. No correlation with HLA phenotype (including Class II DR5 specificity, referred as associated with Hashimoto's thyroiditis, and DQ alleles) was observed.

Sera obtained from planned immunizations between unrelated donors and recipients, identical or compatible at HLA-A and B, were assessed for their capacity to alter the in vitro response of a test panel of lymphocytes to PHA and a purified streptococcal antigen (PAS). In the case of PHA, no serum effects were apparent. The response to PAS, however, significantly inhibited by two sera. When tested for their complement-dependent cytotoxicity on enriched populations of T and B lymphocytes, none of the sera manifested cytotoxicity against T cells nor did serological inhibition correlate with the capacity to lyze B cells. The data suggest that inhibition of the PSA response is mediated by blocking antibodies specific for a subset of lymphocytes, possibly T cells. While the precise mechanism governing the response to PSA is not known, the data are compatible with the idea that an HLA-linked Ir gene, expressed on a subset of T lymphocytes, controls immune responsiveness to PSA.

Peripheral lymphocytes from a panel of individuals who had been assayed for DR specificities by the conventional cytotoxicity assay were typed for DR "supertypic" specificities, DC1 and BR4 x 7, by the radioimmunoassay. A positive and a negative population were clearly distinguished for both specificities and the strong association of the DC1 specificity with DR1, 2, and w6 was confirmed as well as the BR4 x 7 specificity with DR4 and 7. Family study also supported this strong association. Appropriate papain digestion separated molecules carrying DC1 determinant from those carrying DR2 as well as from those carrying DRw6, and separated molecules carrying BR4 x 7 from those carrying DR4. Specificity analysis of the 8th Workshop antisera by use of these separated antigen preparations showed that some anti-DC1 antisera do not possess appreciable anti-DR1, 2, or w6 activity and vice versa. The same was found for DR4 x 7 in its relationship with DR4 and 7. The existing evidence could be explained most economically by assuming a genetic model of two loci in linkage disequilibrium each coding for analogous but distinct forms of the small (beta) subunits of Ia molecules.

The relationship between preoperative CEA, Dukes staging and disease recurrence, was analyzed in 92 patients with colon-rectal cancer, all who underwent curative surgery. Sixty-five of the 92 patients were followed for 36 months. A significant increase in disease recurrence risk is observable starting from a preoperative CEA value of greater than 7.5 ng/ml; corresponding values as such are verified by a significant fall in the actuarial survival curve in comparison to the progress of the curves of the other two groups with lower CEA values. A statistically significant correlation between preoperative CEA and staging was not observed, while both parameters result statistically very reliable (p less than 0.001) for prognosis; preoperative CEA values, less or greater than 7.5 ng/ml can help to stratify the Dukes tumours with respect to the probability of recurrence.

The polymorphism of HLA-DR antigens has been studied by Southern blot hybridization under conditions specific for the detection of the DR beta chain genes. Haplotype-specific patterns were defined with DNA from DR1, 2, 3, 4, 7, w8, w11, w12, and W13 homozygous typing cells, with restriction enzymes Eco RI, Bgl I, and Pvu II. Certain serological specificities, such as DR2, DR3, and DR7, can be encoded by distinct allelic forms of DR beta chain genes. The procedure of "DNA typing" was applied to family analysis of individuals expressing only a single DR specificity upon serological typing. Three cases are described here: (1) in family GR, phenotypic DR 7 homozygotes correspond to genomic heterozygotes, and a novel DR7 allele is described: (2) in family RU, the genes corresponding to a serologically undetected (blank) DR allele were identified by restriction fragment length polymorphism (RFLP); this novel DR haplotype has an RFLP pattern similar to those of the DRw52 family, even though this specificity was not expressed on the DR-blank lymphocytes; (3) in family RG, there is no blank allele, but a homozygote RFLP situation at the DR subregion.

Polymorphism of mitochondrial DNA has been studied in two highland (Desulo, Tonara) and in two lowland (Galtellì, Orosei) Sardinian isolates, formerly subjected to different selective pressure due to malaria, and in 103 individuals from Northern Italy (Bergamo area), where malaria never appeared to be endemic. Two mitochondrial restriction endonuclease patterns (morphs) never described before have been found, one in the Bergamo and Orosei samples, and the other one only in Orosei. Four new mitochondrial types (mitotypes) due to different combinations of morphs have been identified; two of them have been found only in Sardinia, but with such a low frequency that they cannot be defined as typical Sardinian mitotypes. One mitotype (BamHI-morph 3, MspI-morph 4, AvaII-morph 9 and HaeII-morph 1) showed a significantly higher frequency in the highland rather than in the lowland Sardinian villages or in the Bergamo area. Since this mitotype has been found at a relatively high frequency in Central and Southern Italy, while it has been reported to be rare in Caucasians of Central European origin and absent in other ethnic groups (Africans, Chinese, Japanese and Israeli Jews), we suggest it may represent an ancient Mediterranean type. The analysis of these data suggests that drift or other evolutive forces different from malaria might be the major cause of mitochondrial DNA variation in Sardinia.