Multidisciplinary model for hospital-territory integrated management of patient with bone fragility: primary and secondary prevention of fractures according to severity and complexity.

The aim of this study was to promote the construction of a real network and a shared diagnostic and therapeutic management model between hospitals and out-of-hospital healthcare services to capture as many patients with bone fragility as possible. Starting from the analysis of the clinical competences present in the province of Pavia, the bone specialists (BSs) organized some educational events involving both general practitioners (GPs) and hospital specialists. The Fracture Liaison Service (FLS) model, the revision of Note 79, the national plan for chronicity and the health reform of the Lombardy Regional Authority supported the structure of our model, in which the roles of clinicians are well defined and based on the complexity and severity of patients. In our method the GP has a central role as clinical manager, facilitating patient management and communication between the specialists and the BS. In January 2019, the Therapeutic Care Diagnostic Path (PDTA) shared between 2 bone specialists (BSs), 9 GPs, as reference treaters, and a multidisciplinary group of 25 specialists of the Province of Pavia was defined. The strategic directions of the two largest public hospitals in Pavia have supported the PDTA, which was validated by the quality departments of the hospitals themselves. Finally, sixty GPs belonging to the network have joined the PDTA. This model is the first example of integrated management between hospitals and out-of-hospital healthcare services for the primary and secondary prevention of fragility fractures (FF), where the GPs play a pivotal role as managers and supervisors to ensure proper care to chronic patients according to their levels of severity.

Detection of Merkel cell virus and correlation with histologic presence of Merkel cell carcinoma in sentinel lymph nodes.

BACKGROUND: Adjuvant treatment can dramatically improve the survival of patients with metastatic Merkel cell carcinoma (MCC), making early, accurate detection of nodal disease critical. The purpose of this study was to correlate Merkel cell virus (MCV) detection with histopathologic disease in sentinel lymph nodes (SLNs) of MCC. METHODS: Merkel cell carcinoma cases with SLN (n=25) were compared with negative controls (n=27). Viral load was obtained by quantitative polymerase chain reaction (PCR) for regions VP1 and LT3 of MCV. Histopathologic disease and viral load were correlated. RESULTS: Merkel cell virus was detected in 16 out of 17 (94%) of primary MCC (mean viral load (MVL)=1.44 copies per genome). Viral load in the negative controls was <0.01 copies per genome. Merkel cell carcinoma was present in 5 out of 25 (20%) SLN by histopathology, and MCV was detected in 11 out of 25 (44%) MCC SLN (MVL=1.68 copies per genome). In all, 15 out of 25 (60%) SLN showed correlation between histologic and MCV results. In all, 2 out of 25 (8%) samples were histopathologically positive and PCR negative. Of note, 8 out of 25 (32%) samples had detectable MCV without microscopic disease. CONCLUSION: Patients with positive SLN for MCV even if negative by histopathology were identified. The application of molecular techniques to detect subhistologic disease in SLN of MCC patients may identify a subset of patients who would benefit from adjuvant nodal treatment.

DNA methylation profiles delineate epigenetic heterogeneity in seminoma and non-seminoma.

BACKGROUND: It remains important to understand the biology and identify biomarkers for less studied cancers like testicular cancer. The purpose of this study was to determine the methylation frequency of several cancer-related genes in different histological types of testicular cancer and normal testis tissues (NT). METHODS: DNA was isolated from 43 seminomas (SEs), 14 non-SEs (NSEs) and 23 NT, and was assayed for promoter methylation status of 15 genes by quantitative methylation-specific PCR. The methylation status was evaluated for an association with cancer, and between SEs and NSEs. RESULTS: We found differential methylation pattern in SEs and NSEs. MGMT, VGF, ER-ß and FKBP4 were predominately methylated in NSEs compared with SEs. APC and hMLH1 are shown to be significantly more methylated in both subtypes in comparison with NT. When combining APC, hMLH1, ER-ß and FKBP4, it is possible to identify 86% of the NSEs, whereas only 7% of the SEs. CONCLUSIONS: Our results indicate that the methylation profile of cancer-associated genes in testicular cancer correlates with histological types and show cancer-specific pattern for certain genes. Further methylation analysis, in a larger cohort is needed to elucidate their role in testicular cancer development and potential for therapy, early detection and disease monitoring.

Structural characterization of antiidiotypic antibodies. Evidence that Ab2s are derived from the germline differently than Ab1s.

We have found that syngeneic Ab2s in the antiarsonate system are serologically and structurally similar to one another. In contrast, the allogeneic Ab2 response is heterogeneous and derives from a large number of unrelated germline gene segments. The Ab2 response of the BALB/c strain to polyclonal A/J Ars A molecules can probably best be compared with a response to a foreign protein and might have been predicted in a strain that completely lacks the H chain V region gene from which the Ab1 derives. Partial variable region sequences of Ab2s from three other systems in addition to previously reported Ab2 structures indicates that this difference in allogeneic vs. syngeneic Ab2s may be a general phenomena. These data support Jerne's hypothesis of complementary V region genes existing in the germline. However, there is good evidence that these antiidiotypic antibodies are not derived directly from the germline, as somatic processes most likely play an important role in their generation. The D segments of Ab2s in the arsonate system as well as in other systems, are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion may play a role in the generation of the third hypervariable region in these antibodies.

The influence of V kappa gene polymorphism on the induction of silent idiotypes in the arsonate system.

It has been previously shown that it is possible to modify the expressed repertoire of a given individual using idiotypic manipulation. For example, A/J mice respond to arsonate challenge by synthesizing a dominant idiotype, CRIA, whereas BALB/c mice do not. However, after treatment with rabbit polyclonal anti-CRIA antibodies (Ab2 or anti-idiotypic antibodies) and arsonate, BALB/c mice are able to synthesize a CRIA-like idiotype. To determine whether this modification of repertoire is dependent on the immunoglobulin loci (Ig-h, kappa), we have analyzed the anti-arsonate response after anti-idiotypic treatment of three strains of mice (C58, C.C58, AKR), chosen because they are among a small group of strains which express Kappa V regions not seen in other strains. There are also L chains lacking in these strains which are expressed in other mice. The C58 and C.C58 strains share the same Ig-h locus (Ig-ha) with BALB/c mice but C.C58 are congenic mice, that express the kappa loci on a BALB/c genetic background. AKR mice express the Ig-hd haplotype. AKR, C58 and C.C58 do not produce CRIA positive antibodies in response to arsonate; a defect which has been previously mapped to the kappa locus. These three strains of mice (C58, C.C58 and AKR) were treated with rabbit anti-CRIA and boosted with Ars-KLH. The results show that after such treatment, the C.C58 mice were able to express CRIA-like antibodies which are serologically identical to those of BALB/c.

Mutant p53(R175H) upregulates Twist1 expression and promotes epithelial-mesenchymal transition in immortalized prostate cells.

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53(R175H) mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53(R175H), was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53(R175H) mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53(R175H) mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.

Assessment of a functional role of auto-anti-idiotypes in idiotype dominance.

We have used a well-defined idiotypic system, the cross-reactive idiotype of A strain (CRIA) (Ab1) idiotype generated in A/J mice injected with arsonate coupled to keyhole limpet hemocyanin (ARS-KLH), to determine the frequency of precursors for auto-anti-idiotypic antibodies (auto-Ab2) in naive and immunized A/J mice by limiting dilution analysis after polyclonal activation by lipopolysaccharide. In naive animals, the precursor frequencies of auto-Ab2 B cells were below the limit of sensitivity of the technique in the majority of A/J mice, and could be detected in only 20% of the animals. Upon immunization with ARS-KLH, a large increase in auto-Ab2 precursor frequency was observed. This shift in frequency was not found when A/J mice were injected with KLH alone, or when BALB/c mice, which do not express the CRIA idiotype, were injected with ARS-KLH. To study the functional role of the auto-Ab2 B cells, we injected neonatal A/J mice with polyclonal rabbit Ab3 antibodies directed against a recurrent idiotype of auto-Ab2. Thereafter, these mice were injected with ARS-KLH. Although the anti-arsonate response level was normal, the CRIA Ab1 expression was reduced tenfold. Thus, the suppression of auto-Ab2 affects Ab1 dominance. We further show that the presence of maternal Ab1 can strongly modify the immune response of the offspring by inducing higher levels of the idiotype after immunization. Furthermore, IgM anti-arsonate antibodies were detected before immunization with antigen. From these data, we conclude that the affinity of antigen alone cannot explain the dominance of CRIA. Network selection is important in the shaping of the available repertoire.

Molecular characterization of monoclonal CRIA-positive anti-arsonate antibodies derived from idiotype-negative mice bearing a light chain polymorphism.

We have elicited anti-arsonate antibodies bearing the major cross-reactive idiotype (CRIA) in a double congenic idiotype-negative strain (C.C58.AL-20) bearing a light chain polymorphism that has previously been shown serologically not to complement idiotype-positive heavy chains. Using the idiotype cascade (Ab1-->Ab2-->Ab3-->-->Ab1'), CRIA-positive antibodies were raised and monoclonal antibodies were isolated and characterized serologically and by nucleotide sequence analysis. Two types of idiotype-positive anti-arsonate antibodies were generated in the C.C58.AL-20 strain. One group of hybridomas used the canonical VH1.8 heavy chain gene segment with V kappa 10 variant light chains. A second group used a VHGAM3.8 heavy chain with V kappa 10 variant light chains. This latter heavy-light pairing has been observed in CRIA-like responses previously in BALB/c mice after idiotypic manipulation (or rarely after antigen alone). These studies demonstrate the plasticity of the immune response when manipulated with idiotype reagents as well as its structural variability. Additionally, they provide important insights into the potentials of idiotype vaccines.

The perinatal presence of antigen (p-azophenylarsonate) or anti-mu antibodies lead to the loss of the recurrent idiotype (CRIA) in A/J mice.

The immune response of A/J mice against p-azophenylarsonate (Ars)-keyhole limpet hemocyanin (KLH) is characterized by the dominance, late in primary and during the secondary, of a recurrent idiotype called CRIA, encoded by a canonical combination of Ig gene segments. In this study, A/J mice were given Ars coupled to deaggregated human gamma globulins (dHGG) within 24 h after delivery. The offsprings from these mice were then exposed as adults to Ars-KLH. These animals developed an unusual immune response. The level of anti-Ars antibodies was nearly normal but a dramatic shift in repertoire was observed: the cross-reactive idiotype which is the hallmark of the anti-Ars response in A/J mice was completely absent. The idiotype could be recovered by injection of anti-idiotypic antibodies alone, with no need of lipopolysaccharide coupling. Therefore the presence of antigen at birth can lead to a strong perturbation of idiotype selection. Similar results were obtained with neonatal treatment using anti-IgM antibodies. After recovery of suppression, A/J mice can mount an anti-arsonate response of normal level but devoid of the dominant idiotype.

Molecular and cellular basis of the altered immune response against arsonate in irradiated A/J mice autologously reconstituted.

The humoral immune response to arsonate (Ars) in normal A/J mice is dominated in the late primary and particularly in the secondary response by a recurrent and dominant idiotype (CRIA) which is encoded by a single canonical combination of the variable gene segments: VHidcr11-DFL16.1-JH2 and Vkappa10-Jkappa1. Accumulation of somatic mutations within cells expressing this canonical combination or some less frequent Ig rearrangements results in the generation of high-affinity antibodies. By contrast, in partially shielded and irradiated A/J mice (autologous reconstitution) immunized with Ars-keyhole limpet hemocyanin (KLH), both the dominance of the CRIA idiotype and the affinity maturation are lost, whereas the anti-Ars antibody titer is not affected. To understand these alterations, we have analyzed a collection of 27 different anti-Ars hybridomas from nine partially shielded and irradiated A/J mice that had been immunized twice with Ars-KLH. Sequence analysis of the productively rearranged heavy chain variable region genes from those hybridomas revealed that (i) the canonical V(D)J combination was rare, (ii) the pattern of V(D)J gene usage rather corresponded to a primary repertoire with multiple gene combinations and (iii) the frequency of somatic mutations was low when compared to a normal secondary response to Ars. In addition, immunohistological analysis has shown a delay of 2 weeks in the appearance of full blown splenic germinal centers in autoreconstituting mice, as compared to controls. Such a model could be useful to understand the immunological defects found in patients transplanted with bone marrow.

A private idiotype can become recurrent through genetic recombination and gene(s) unlinked to the Igh locus governs its expression.

Any immune response is characterized by its idiotypic profile. Two different kinds of idiotype (Id) have been described. Private Id are restricted to a few individuals from a species while recurrent Id appear in a large majority of individuals from the same species immunized with the same antigen. We describe, in this report, an experimental model whereby a private Id can become recurrent through genetic recombination. The immune response of A mice against the hapten arsonate is characterized by a recurrent Id called cross-reactive idiotype A (CRIA). A strongly CRI, called CRIA-like, can be occasionally detected in some BALB/c mice (5% to 10%) immunized with arsonate. Molecular studies show that CRIA and CRIA-like antibodies have highly homologous D segments and identical light chains. By contrast, their VH segments are vastly dissimilar. We have examined the anti-arsonate response of inbred strains of mice whose Igh loci are recombinant between those of A/He and BALB/c. Interestingly, we have observed that the CRIA-like Id which is private in BALB/c becomes recurrent in the AXC-1 strain which harbors the VH genes from BALB/c, the DH and CH genes from A/He. Structural studies demonstrate that highly homologous, VH, VL and D segments are used in BALB/c and AXC-1 mice. The basis for this differential expression of highly similar genes could be linked to the DH locus. However, F1 mice stemming from the cross between AXC-1 and BALB/c do not express the Id. The backcross analysis shows that the non-expression of the Id in F1 mice depends on genes unlinked to the Igh locus.

Distinct VH repertoires in primary and secondary B cell lymphocyte subsets in the preimmune repertoire of A/J mice: the CRI-A idiotype is preferentially associated with the HSA(low) B cell subset.

The anti-arsonate immune response of A/J mice is characterized by the occurrence of several recurrent idiotypes with a different temporal pattern of expression. The CRI-A idiotype is typically a memory idiotype since it appears late in the primary and dominates the secondary as well as subsequent immune responses. The CRI-C idiotype is present throughout the responses, including the primary one. Naive adult A/J mice treated repeatedly with anti-mu or anti-delta monoclonal antibodies exhibit a completely different balance of HSA(low) and HSA(high) B cell subsets and an opposite idiotype profile after immunization with p-azophenylarsonate coupled to hemocyanin. Anti-mu treatment leads to a striking enhancement of the HSA(low) cell subset associated with an earlier important synthesis of CRI-A(+) antibodies, while anti-delta treatment enhances significantly the HSA(high) compartment with a strong decrease of CRI-A and persistence of CRI-C1 antibodies. Semiquantitative PCR analysis reveals that the presence of CRI-A transcripts is associated with the HSA(low) compartment, while CRI-C transcripts are mainly associated with HSA(high) B cell subsets. This has been demonstrated with spleen cells of adult A/J mice treated with anti-mu or anti-delta antibodies and also with purified B cell subsets of unimmunized adult A/J mice and on neonatal spleen cells. It appears that the memory (CRI-A) idiotype is selected into the HSA(low) B cell subset before antigen arrival.