Clonal evolution of lung tumors.

Lung tumors, particularly squamous cell carcinomas, are believed to develop through a series of morphological abnormalities, driven by underlying somatic genetic changes. One way of studying this process is to analyze candidate somatic genetic changes in samples of squamous metaplasia and bronchial dysplasia of varying degrees of severity as well as tumor from the same patient. This assumes a clonal relationship between these lesions. In this article, we provide evidence that adjacent, physically distinct bronchial abnormalities are clonally related. This has been achieved using a plaque assay technique to detect the same p53 mutation, present throughout a tumor specimen, in a small proportion of cells in an adjacent squamous metaplasia. In addition, we have obtained two dysplasia samples from a tumor-free patient over a 9-month interval. The earlier sample had one p53 mutation, whereas the later sample has to p53 mutations on different alleles. Thus, the pattern of clonal evolution detected in the parallel samples mimics the pattern seen in longitudinal samples and supports the analysis of synchronously collected samples for the study of tumor progression.

p53 and chromosome 3 abnormalities, characteristic of malignant lung tumours, are detectable in preinvasive lesions of the bronchus.

Bronchial epithelial dysplasia is believed to precede invasive squamous cell carcinoma of the lung. Six paired dysplasia and tumour samples were distinguished histologically in sections of formalin-fixed paraffin-embedded lung tissue from patients with lung cancer. Additionally, samples of dysplastic bronchial epithelium were obtained from patients without lung tumours. Microdissection of the unstained sections provided dysplastic and tumour samples from which DNA was prepared for comparison with the patients' constitutional genotype, using polymerase chain reaction-based restriction fragment length polymorphism analysis. All six samples of tumour and the paired adjacent samples of bronchial dysplasia showed loss of heterozygosity (LOH) at loci on the short arm of chromosome 3. Five of the six cases showed involvement of the p53 gene as assessed by LOH at the AccII site within the gene, and by immunoreactivity to CM-1, an antibody which recognizes the mutated form of the p53 protein in paraffin-embedded material. Of the dysplastic samples, obtained from patients without invasive tumours, all three showed LOH at 3p; one sample showed LOH at the AccII polymorphic locus within the p53 gene, and another sample, uninformative at this locus, stained positively with this antibody. These results indicate that somatic genetic changes are present in preinvasive lesions in the bronchus.

Sequential molecular genetic changes in lung cancer development.

Epithelial tumours develop through a sequence of pre-invasive lesions of increasing disarray driven by underlying somatic genetic changes. We have studied the occurrence of the two most common somatic genetic changes associated with lung cancer in a series of premalignant bronchial lesions representing different stages in lung tumorigenesis. We present evidence that allele loss on chromosome 3 precedes damage to the p53 gene. Damage to chromosome 3 itself appears to be sequential in that the pattern of allele loss seen in dysplasia is often much more discrete than in invasive tumours. This implies that preneoplastic lesions may be a useful source of material for deletion mapping studies aimed at localising the position of tumour suppressor genes. We illustrate this by the comparison of an interstitial deletion described in this study with a homozygous deletion we have described previously, which has resulted in a better definition of the localisation of a tumour suppressor gene believed to be involved in lung cancer development.

Increased elastin content and decreased elastin concentration may be predisposing factors in dissecting aneurysms of human thoracic aorta.

OBJECTIVE: The aim was to investigate whether changes in elastin distribution in the thoracic aorta are associated with occurrence of dissecting aneurysms. METHODS: Ten thoracic aortas were obtained at necropsy from dissections (mean patient age 74.3 years, SD 7.3) and from 10 age matched controls (mean age 73.1 years, SD 6.9). Full wall thickness samples (1 cm diameter) were taken at 12 sites between heart and diaphragm from aortas of dissections and controls. Elastin content (total elastin per sample), concentration (mg.mg-1 tissue dry weight), degree of cross linking, and amino acid composition were determined. RESULTS: Comparison of areas of dissected aortas involved in dissection with corresponding areas of controls showed significant increases in content of elastin (p < 0.05), content and concentration of proteins other than elastin and collagen (p < 0.01), and a decrease in elastin concentration (p < 0.01). Comparison of areas remote from dissection with corresponding areas in controls showed no significant differences except for decreased elastin concentration (p < 0.05). There were no differences in elastin cross linking. Elastin from dissected aortas had a higher content of aspartate, threonine, serine, glutamate, and lysine and a lower content of glycine, alanine, and valine than elastin from controls (p < 0.05). CONCLUSIONS: Biochemical changes in dissections are localised to the dissected area, with increased deposition of elastin, collagen, and other proteins. The altered matrix composition is likely to change the mechanical properties, possibly increasing the tendency to rupture.

Biochemical investigation of possible lesions in human aorta that predispose to dissecting aneurysms: pyridinoline crosslinks.

Pyridinoline, a collagen specific covalent crosslink, was quantified in acid hydrolysates of human aorta using a non-equilibrium inhibition ELISA. The study was based on specimens from seven cases of aortic dissection and from seven control subjects whose death was unrelated to thoracic aortic dissection. There were no significant differences in the amounts or concentrations of pyridinoline in aortas with dissecting aneurysms compared with normal tissue, thus excluding the possibility of a causative relation between the degree of pyridinoline crosslinking of collagen molecules and dissection of the thoracic aorta. In all cases, however, the number of pyridinoline crosslinks per molecule of collagen in the ascending aorta and arch approached the theoretical maximum for lysyl derivatives and was as high as that present in cartilage. Thus in this region of the vessel pyridinoline represents the major stabilising crosslink of collagen. In contrast, the number of pyridinoline crosslinks per collagen molecule decreased maximally by a factor of 10 between the arch and the proximal regions of the descending thoracic aorta. This suggests a possible correlation between the rigidity of collagen fibres and the forces exerted on the aortic wall during diastole and systole.

TNF alpha mRNA and protein in cardiac transplant biopsies: comparison with serum TNF alpha levels.

OBJECTIVES: To examine the role of TNF alpha (TNF alpha) in cardiac transplant rejection by simultaneous analysis of protein expression and its messenger RNA within serum and grafted tissue. METHODS: 54 endomyocardial biopsy specimens were taken from 19 patients at various times after transplantation. TNF alpha messenger RNA was localised using a digoxygenin labelled complementary DNA probe. An anti-TNF alpha antibody was used to immunohistochemically label the protein product. Serum TNF alpha levels at the time of biopsy were analysed using a specific enzyme-linked immunosorbent assay. RESULTS: TNF alpha mRNA was present in 22/34 endomyocardial biopsies. Eight also contained TNF alpha protein. None had protein alone. Expression did not relate to the grade of rejection in the present or subsequent biopsies. Serum TNF alpha was undetectable (assay sensitivity 30-330 pg/ml) for the majority of specimens. In the nine cases with elevated serum levels, eight samples were from cases within the first 30 days post transplant (r = -0.379; P < 0.05). CONCLUSIONS: Neither tissue TNF alpha mRNA, tissue protein, nor serum TNF alpha relate to the grade of rejection. Furthermore, TNF alpha expression within endomyocardial biopsies is not reflected in the serum. These findings argue against the use of serum analysis as an indicator of cytokine profiles within cardiac tissue allografts. The demonstration of a trend in the early expression of TNF alpha after transplantation suggests that its release may not be specific to the process of rejection.

Mesotheliomas with deciduoid morphology: a morphologic spectrum and a variant not confined to young females.

Deciduoid mesotheliomas are rare with only four previously reported cases, all affecting the peritoneum of young females. We describe another six cases (three men and three women; age range 52-65 yrs, median 55 yrs; five peritoneal and one pleural). Three patients had an occupational history of asbestos exposure. The deciduoid appearance predominated in four cases, whereas in two it represented a minor component within conventional tubulopapillary epithelioid mesothelioma. All tumors were strongly cytokeratin-positive (including CK5/6) and all showed at least focal staining for thrombomodulin, HBME-1, and calretinin. All were negative for epithelial mucin (D/PAS), CEA, BerEP4, LeuM1 (CD15), CD21, CD35, and S100 protein. Five of six cases (83%) were vimentin-positive and two (33%) were focally positive for alpha-smooth muscle actin. A differential diagnosis of gastrointestinal autonomic nerve tumor (GANT) had been initially considered from the morphology of one case, and we found positivity for some of the "neuronal" markers described in GANTs. This prompted us to apply such a panel to the other five tumors, accepting that the cytokeratin positivity encountered in all of our cases would exclude GANT. All cases of deciduoid mesothelioma (100%) were positive for PGP 9.5 and NSE and four of six (66%) were positive for NKI/C3. Weak focal staining (<5% cells) for synaptophysin was seen in two of six tumors. All cases were chromogranin-negative. All cases examined by electron microscopy showed desmosomes and smooth microvilli without rootlets but no neuroendocrine granules. In conclusion, a deciduoid morphology appears to be part of the histopathologic spectrum encountered in epithelioid mesothelioma. This variant is not confined to female patients and occurs over a wider age range than previously recognized. The overlapping immunophenotype with GANTs illustrates that caution should be exercised when interpreting positivity for "neuronal" markers in this context. An immunohistochemical panel that includes cytokeratins should always be used.

Hypertonic citrate solution as an alternative to modified Euro-Collins' solution for lung preservation.

In a canine model of acute ischemic lung injury, a hypertonic citrate solution (HTC) widely used for renal preservation in the United Kingdom, was compared with modified Euro-Collins' solution (ECS) currently the most widely clinically used pulmonary perfusate. Ten beagle dogs underwent left thoracotomy and exclusion of the left lung in situ. The lung was flushed with 30 ml/kg of either HTC or ECS and subjected to 60 min of warm ischemia. The circulation to the lung was then restored, the contralateral lung excluded, and the animal ventilated at a fixed FiO2 of 0.4 for 4 hr. Lung function was assessed by arterial oxygenation and hemodynamic measurements and, following sacrifice, by lung weight gain, bronchoalveolar lavage, and ultrastructural studies. Flush perfusion with HTC was associated with significantly less severe reperfusion injury, as determined by superior arterial oxygenation (PaO2 at 1 hr: HTC--152 mmHg [(95% confidence interval) CI] [122-182], ECS--59 [47-70]; PaO2 at 4 hr: HTC--124 [100-149], ECS--51 [42-61]), lower pulmonary vascular resistance index (PVRI at 4 hrs: HTC--838 dynes sec cm-5m-2 [651-1075], ECS--1233 [963-1588]); and lower lung weight (HTC--85 g [66-107], ECS--146 [114-184]). Bronchoalveolar lavage studies demonstrated an influx of neutrophils following reperfusion that was significantly less marked in the HTC group (increase in % neutrophils: HTC 24 [19-29], ECS 77 [72-82]). Lung injury assessed by electron microscopy tended to be less severe in the HTC animals. We conclude that HTC may offer an alternative superior to ECS for lung preservation.

Genotypic variation in the transforming growth factor-beta1 gene: association with transforming growth factor-beta1 production, fibrotic lung disease, and graft fibrosis after lung transplantation.

BACKGROUND: Transforming growth factor (TGF)-beta1 is a profibrogenetic cytokine that has been implicated in the development of fibrosis in transplanted tissues. In this study, we have analyzed the genetic regulation of TGF-beta1 production in lung transplant recipients. METHOD: A polymerase chain reaction-single-stranded conformational polymorphism technique was used to detect polymorphisms in the TGF-beta1 gene from genomic DNA. Polymorphisms were shown to correlate with in vitro TGF-beta1 production by stimulated lymphocytes. A single-specific oligonucleotide probe hybridization method was devised to screen for these polymorphisms in lung transplant groups and controls. RESULTS: We have identified five polymorphisms in the TGF-beta1 gene: two in the promoter region at positions -800 and -509, one at position +72 in a nontranslated region, and two in the signal sequence at positions +869 and +915. The polymorphism at position +915 in the signal sequence, which changes codon 25 (arginine-->proline), is associated with interindividual variation in levels of TGF-beta1 production. Stimulated lymphocytes of homozygous genotype (arginine/arginine) from control individuals produced significantly more TGF-beta1 in vitro (10037+/-745 pg/ml) compared with heterozygous (arginine/proline) individuals (6729+/-883 pg/ml; P<0.02). In patients requiring lung transplantation for a fibrotic lung condition, there was an increase in the frequency of the high-producer TGF-beta1 allele (arginine). This allele was significantly associated with pretransplant fibrotic pathology (P<0.02) (n=45) when compared with controls (n=107) and with pretransplant nonfibrotic pathology (P<0.004) (n=50). This allele was also associated with allograft fibrosis in transbronchial biopsies when compared with controls (P<0.03) and with nonallograft fibrosis (P<0.01). CONCLUSION: The production of TGF-beta1 is under genetic control, and this in turn influences the development of lung fibrosis. Hence, the TGF-beta1 genotype has prognostic significance in transplant recipients.

Rejection in heart transplantation strongly correlates with HLA-DR antigen mismatch.

It is well established that incompatible HLA antigens presented by donor tissue readily evoke an immune response. Prospective HLA matching policies, widespread in European kidney transplant centers have reduced the level of HLA mismatching and have significantly improved graft survival. The influence of HLA incompatibility in heart transplantation remains controversial, and prospective HLA matching is seldom achieved. We examined the role of HLA antigen mismatching on transplant rejection by analyzing 2569 endomyocardial biopsies (EMB) from 157 consecutive orthotopic heart transplants performed from April 1987 to August 1993 in our own center. Biopsies were graded according to the accepted International Classification, with grade 2 and higher indicating rejection. Among 91 patients who received a 2 HLA-DR mismatch transplant 34% of 1624 biopsies analyzed were graded as > or = 2. This frequency fell to 29% of 797 biopsies for 53 patients with a one-HLA-DR mismatch and to 18% of 148 biopsies for 13 patients in the zero-HLA-DR-mismatch group (P < 0.00005). No significant effect on EMB grade frequencies was observed using the same method of analysis with transplants mismatched at the HLA-A or HLA-B loci apart from analysis of HLA-B matched transplants at 3 months posttransplant (P = 0.02). The close linkage of the HLA-B and HLA-DR loci may account for this observation. The results of this study show that heart transplants matched at the HLA-DR locus have a significantly reduced incidence of EMB grades indicative of rejection requiring augmented immunosuppressive therapy. We propose that prospective HLA-DR matching should be adopted for allocation of donor hearts for more efficient use of this precious and limited resource.

The influence of histocompatibility on graft rejection and graft survival within a single center population of heart transplant recipients.

BACKGROUND: We report a consecutive single center series of 261 patients who received first orthotopic heart transplants from 1986 to 1997. The 1- and 5-year graft survivals were 78 and 68%. The influence of histocompatibility was investigated by comparing graft survival and numbers of treated rejection episodes with HLA-A, -B, and -DR mismatches over different time periods. FINDINGS: Recipients with six mismatches for HLA-A+-B+-DR combined (13.4%) had reduced survival at 7 years (47%) when compared with other recipients (64%). In the first year of transplant, recipients with four HLA-A+-B mismatches had significantly reduced actuarial graft survival (P=0.03) with the greatest influence apparent at 6 months [0-3 mismatches (n=193) 85% versus 4 mismatches (n=68) 69%; P=0.005, OR=2.1]. For 182 recipients with functioning hearts at 1 year, the number of rejection episodes treated within this time was strongly influenced by HLA-DR mismatch [0 DR mismatch (n=15) mean 1.2 rejection episodes versus 1 DR mismatch (n=76) mean 2.7 rejection episodes versus 2 DR mismatches (n=91) mean 3.8 rejection episodes: P=0.0002]. Of these 182 transplants, recipients who had more than four treated rejection episodes during the first year had a significantly reduced 7- year survival [<5 rejection episodes (n=133) 85% versus more than four rejection episodes (n=49) 66%; P=0.02, OR=3.4], as did those with two HLA-DR mismatches [0+1 mismatch (n=91) 87% versus 2 mismatches (n=91) 70%; P<0.05, OR=2.4]. INTERPRETATION: We show that graft loss in the first 6 months of transplant is significantly influenced by four HLA-A+-B mismatches. HLA-DR mismatch significantly increases the number of rejection episodes within the first year, without influencing graft survival. After 12 months both >4 rejection episodes in the first year and two HLA-DR mismatches are markers for late graft loss. We postulate that immunological graft loss in the first 6 months is dominated by the direct allorecognition pathway driven by HLA-DR mismatch. This mechanism is later lost or suppressed. Our data highlight HLA-DR mismatch as a marker for late graft loss and we show an advantage to avoiding transplanting hearts with six HLA-A+-B+-DR mismatches and to minimizing HLA-DR mismatches whenever possible.

Amelioration of lung ischemic injury with prostacyclin.

The single-flush technique of lung preservation is thought to be enhanced by prostaglandin treatment. In order to test this hypothesis, ten beagle dogs underwent thoracotomy and in situ flush perfusion of the excluded left lung with 30 ml/kg of cold, modified Euro-Collins' solution. Group 1 (n = 5) received pretreatment with 30 ng/kg/min of PGI2 by infusion and as an additive to the flush (20 micrograms/L). Group 2 (n = 5) received no PGI2 and served as controls. Following 60 min of warm ischemia, the left lung was reperfused, the contralateral lung excluded, and the animal ventilated at a fixed FiO2 of 0.4 for 4 hr. The severity of reperfusion injury was assessed by arterial oxygenation and hemodynamic measurements and, following sacrifice, by lung weight gain and bronchoalveolar lavage and ultrastructural studies. PGI2 therapy resulted in significant amelioration of reperfusion injury, with superior oxygenation at both 1 and 4 hr (PaO2 at 1 and 4 hr, respectively; PGI2: 145 mmHg +/- 17.0 and 114 +/- 11.2; no PGI2: 59 mmHg +/- 5.8 and 51 +/- 4.5; P less than 0.01 at both times), lower pulmonary vascular resistance index at 4 hr (PVRI; PGI2: 913 dynes sec cm-5m-2 +/- 91; no PGI2: 1239 +/- 68; P less than 0.05) and lower lung weight (PGI2: 76 g +/- 4; no PGI2: 146 +/- 10; P less than 0.001). Bronchoalveolar lavage studies revealed an influx of neutrophils following reperfusion that was less marked in the PGI2 group (increase in % neutrophils; PGI2: 50.4 +/- 6.7; no PGI2: 76.9 +/- 6.0; P less than 0.05). Lung injury score assessed by electron microscopy was lower in the PGI2 group (PGI2: 5.2 +/- 1.1; no PGI2; 8.1 +/- 0.5; P less than 0.05). It is concluded that PGI2 treatment is protective against ischemic lung injury in this model.

Tumor necrosis factor-alpha gene polymorphism and death due to acute cellular rejection in a subgroup of heart transplant recipients.

Irreversible acute rejection of the transplanted heart usually has a fatal outcome. Predicting which recipients are most likely to reject might allow closer monitoring and modification of treatment protocols to prevent graft loss. Recipients genetically predisposed to produce more TNF-alpha are those who suffer the most acute rejection episodes. Here we show that TNF-alpha genotype is strongly associated with death due to acute cell-mediated heart transplant rejection (Chi-square = 28.57, p < 0.0001). This subgroup of recipients should be given optimally tissue matched transplants and should be treated with the most effective immunosuppressive regimens.

Pulmonary atypical carcinoid: predictors of survival in 106 cases.

Pulmonary neuroendocrine tumors (NE) include a spectrum of tumors from typical carcinoid (TC) to atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell carcinoma (SCLC). Little is known about prognostic predictors for AC because of its rarity. Survival analysis was performed on 106 ACs with clinical follow-up from the AFIP and the Pathology Panel of the International Association for the Study of Lung Cancer (IASLC). The tumors fulfilled the 1999 WHO/IASLC criteria for AC of a NE tumor with a mitotic rate of 2 to 10 per 2 mm(2) of viable tumor or coagulative necrosis. Multiple clinical and histologic features were analyzed by Kaplan-Meier and Cox regression analysis. Of the clinical features, higher stage (P = .003) and a tumor size of 3.5 cm or greater (P = .003) were associated with a worse prognosis. Features that were histologically unfavorable by univariate analysis were mitotic rate (P =.002), pleomorphism (P = .018), and aerogenous spread (P =.007). Histologically favorable features by univariate analysis were the presence of palisading (P = .008), papillary (P = .039), pseudoglandular (P =.026), and rosette (P = .022) patterns. Female gender showed a trend toward a poorer prognosis (P =.085) and was included in the multivariate model. Multivariate analysis stratified for stage showed mitoses (P<.001), a tumor size of 3.5 cm or greater (P =.017), and female gender (P =.012) to be the only negative independent predictors of prognosis and the presence of rosettes (P = .016) to be the only independent positive predictor. We further divided the AC into subgroups of low (2 to 5 mitoses/2 mm(2)) and high (6 to 10 mitoses/2 mm(2)) mitotic rate and compared the survival with TC and with LCNEC. Within the category of AC, the patients with a higher mitotic rate had a significantly worse survival than those with a lower mitotic rate (P<.001) stratified for stage. Five- and 10-year survival rates for AC (61% and 35%, respectively) stratified for stage were significantly worse than for TC and better than that for LCNEC and SCLC. Chemotherapy or radiation therapy was given in 12 of 52 and 14 of 52 cases, respectively, but the data were insufficient to evaluate tumor response. We conclude that AC is an aggressive neuroendocrine neoplasm with survival intermediate between TC and LCNEC and SCLC. Higher mitotic rate, tumor size of 3.5 cm or greater, female gender, and presence of rosettes are the only independent predictors of survival. Surgical resection remains the treatment of choice, and the role of chemotherapy and radiation therapy remains to be proven.

Pulmonary diffusion abnormalities in heart transplant recipients. Relationship to cytomegalovirus infection.

Lung function of patients with heart failure is characterized by a variety of changes proposed as being due to passive congestion, secondary pulmonary fibrosis, and/or recurrent pulmonary emboli. A diffusion impairment thought to be due to cyclosporine has also been noted in patients following heart transplantation. Similar changes of unclear origin have been observed in renal transplant recipients. The objective of this study was to determine the extent to which lung function changes are reversible by cardiac transplantation and relate changes to the status of the recipients lung in the presence of possible vascular, iatrogenic, immune, or infectious injury. We analyzed the data of 22 patients who underwent lung function testing before and after heart transplantation and correlated changes to hemodynamic change, episodes of rejection, concentration of cyclosporine, and cytomegalovirus infection. Despite excellent graft function, the carbon monoxide transfer factor deteriorated to a mean of 57 percent of predicted postoperatively. The fall in diffusion factor did not correlate with episodes of cardiac rejection, cyclosporine levels, or hemodynamic status. In those patients who had serologic evidence of cytomegalovirus infection, the reduction in transfer factor was greater compared to those without infection despite a normal chest radiograph. The effects of cardiopulmonary bypass were unlikely to have been responsible for the abnormalities as lung function was assessed at a mean of 14 months after surgery. In heart transplant recipients, a change in diffusion capacity may represent an additional marker for cytomegalovirus infection and reflect infectious/immune injury late following surgery.

Transforming growth factor beta in relation to cardiac allograft vasculopathy after heart transplantation.

BACKGROUND: Cardiac allograft vasculopathy is a frequent sequel to cardiac transplantation, but the role of cytokines on the subsequent development of vasculopathy is still largely unknown. METHODS: We retrospectively studied 172 heart transplant recipients to investigate the relationship between the development of vasculopathy and various factors including the presence of transforming growth factor (TGF-beta) in the graft. Endomyocardial biopsy specimens were stained with antibodies for TGF-beta and CD+68, and a TGF-beta staining score was derived. Vasculopathy was diagnosed by angiography and rejection was graded according to the International Society of Heart and Lung Transplantation classification. TGF-beta(1) genotype was determined by polymerase chain reaction analysis of DNA. RESULTS: After a mean follow-up period of 68 +/- 32 months, the prevalence of significant vasculopathy was 52%. The TGF-beta staining score was higher in patients with more severe vasculopathy (95% confidence interval = 8.9-12.1) than in those who showed minimal or mild vasculopathy score changes of more than 7 (95% confidence interval = 3.4-5.1), P =.0001. TGF-beta expression correlated with the degree of vasculopathy (r = 0.73, P <.0007) during the study period. Risks for vasculopathy were recipient homozygous TGF-beta genotype, recurrent rejection, recipient history of ischemic heart disease, donor male sex, old donor age (years), and donor history of subarachnoid hemorrhage. CONCLUSION: A strong association exists between the expression of TGF-beta in cardiac biopsy specimens and the development of vasculopathy. TGF-beta in the cardiac allograft is related to its genotype and to the number of rejection episodes. Strategies to down-regulate TGF-beta production might improve the outcome of cardiac allografts.

Bone histomorphometry in adult patients with cystic fibrosis.

STUDY OBJECTIVE: Low bone mineral density is a common complication of cystic fibrosis (CF), and recent studies have implicated vitamin D insufficiency as a significant etiologic factor. The aim of this study was to establish whether there was bone biopsy evidence of vitamin D deficiency osteomalacia in patients with CF and to document the general histomorphometric characteristics of CF bone. PATIENTS AND METHODS: A retrospective descriptive and histomorphometric study of postmortem L2/L3 vertebral bone biopsy specimens was undertaken on tissue from 11 posttransplant CF patients and 4 nontransplanted CF patients. Control data were derived from postmortem bone specimens from 15 young adults. RESULTS: Bone from all CF patients was characterized by severe osteopenia in both trabecular and cortical bone. At the cellular level, there was decreased osteoblastic and increased osteoclastic activity. The reduction in osteoblastic activity was due to both a decrease in osteoblast number and a decrease in the biosynthetic potential of osteoblasts. The osteoclastic changes were due to an increase in the number of osteoclasts. The increase in osteoclasis and the uncoupling of osteoblastic and osteoclastic activity resulted in an increase in resorptive surfaces. Although there were few significant differences between the transplanted and nontransplanted CF groups, both cortical and trabecular bone mass tended to be lower after transplantation. None of the CF undecalcified biopsy specimens showed osteoid parameters characteristic of vitamin D deficiency osteomalacia. CONCLUSIONS: CF patients have an unusual and complex pattern of cellular changes within bone that are not typical of vitamin D deficiency osteomalacia.

Interleukin-10 in human heart transplantation: an in situ hybridization study. The Manchester Transplant Cytokine Group.

BACKGROUND: The role of interleukin-10 in graft acceptance and rejection was investigated by analysis of its messenger RNA expression within endomyocardial biopsy material from heart transplant recipients. METHODS: Forty-six biopsy specimens were analyzed from 19 patients (16 male, 3 female), with an age range of 15 to 62 years (mean = 47 years). Biopsy specimens were "snap" frozen in liquid nitrogen, and 10-microns thick sections were cut and postfixed in 4% paraformaldehyde. Messenger RNA for interleukin-10 was localized with nonradioactively (digoxigenin) labeled complementary DNA probes and detected immunoenzymatically with an antidigoxigenin polyclonal antibody. The histopathologic diagnosis of rejection was made according to the criteria of the Heart Rejection Study group. RESULTS: Interleukin-10 transcripts were detected in 12 of 36 rejecting biopsy specimens. None of the ten nonrejecting biopsy specimens were positive. Expression within the rejection infiltrate was more prominent in biopsy specimens from milder rejection episodes. By contrast, in biopsy specimens from moderate rejection, expression was mainly within areas of fibrosis. The expression of interleukin-10 transcripts did not relate to the number of previous rejection episodes nor to the histologic grade of the subsequent biopsy specimens. CONCLUSIONS: This study emphasizes the importance of in situ techniques in localizing cytokine expression in relation to tissue structure and suggests that interleukin-10 may serve a function in the immune regulation of the infiltrate at sites of inflammation, rather than in immune suppression of the rejection process. Further study is necessary to elucidate the precise role of interleukin-10 in transplantation in relation to the overall profile of cytokine expression within the rejection infiltrate.

RANTES chemokine expression is related to acute cardiac cellular rejection and infiltration by CD45RO T-lymphocytes and macrophages.

BACKGROUND: Despite the advances made in immunosuppression therapy, episodes of acute cellular rejection may affect graft function and survival. We investigated the role of RANTES in cellular recruitment and in cardiac allograft rejection. METHODS: Endomyocardial biopsies (n = 65) from 30 patients were taken at various times after transplantation. In 4 subjects who died of acute cellular rejection, the profile of RANTES expression was monitored in all biopsy specimens and in postmortem tissue. Myocardial tissue from 10 other transplants was also analyzed. Sections were stained with an anti-human RANTES antibody with the streptavidin-biotin technique. RANTES-positive cells were related to macrophage, CD45RO "memory" T-cell, and eosinophil infiltration. RESULTS: RANTES-positive cells were identified within the cellular infiltrate in 95% of biopsies with moderate/severe rejection and 28% with mild rejection. RANTES-positive, CD45RO-positive, and macrophage cell numbers were higher in subjects who died of acute cellular rejection than of other causes. A highly significant difference in RANTES-positive cell number was observed between moderate/severe, mild, and nonrejection groups (p = .0001) and correlated significantly with macrophage number in both right and left ventricles (r = .693, p < .01; r = .599, p < .05, respectively) and with the number of "memory" T cells (r = .829, p < .001; r = .779, p < .01, respectively). CONCLUSIONS: These findings suggest that local release of RANTES is important in the recruitment of both macrophages and CD45RO T cells in cardiac allograft rejection. RANTES may be an important chemokine to target for therapeutic intervention in heart rejection.