Pasteurized autogenous bone graft for reconstruction after resection of malignant bone and soft tissue tumors: imaging features.

Our experimental study revealed that pasteurization was sufficient for killing all tumor cells in any massive bone and that the osteoinductivity and mechanical strength of pasteurized bone were about same as that of a fresh bone graft and better than boiled or autoclaved bone. We have performed reconstruction surgery using pasteurized autogenous bone graft since 1990. Resected bone was warmed for 30 minutes in a homeothermal heater set at 60 degrees C and then placed back to the original anatomic site. The graft was fixed with a plate or intramedullary nail with or without bone cement. Based on our experience in 31 patients, no local recurrence was seen in the grafts. Among 24 patients who can be adequately evaluated, incorporation of graft was complete in 17 cases and partial in 4 cases. In 18 of 21 patients, uptake of radionuclide in the grafted pasteurized bone was detected on bone scintigraphy approximately 6 months after surgery, and the uptake increased gradually, which suggested revascularization and remodeling of grafted bone.

Treatment modalities for metastatic bone tumors and associated issues: focusing on surgical indications and techniques for metastatic lesions in limb bones.

Metastatic bone tumors are traditionally the most commonly encountered form of malignant bone tumor. In recent years, the prevalence of metastatic bone tumors has shown a tendency of further increase, due to advances in cancer treatments that prolong survival. Accompanying this increase, the chances that orthopedic surgeons will encounter these tumors and the need for surgical treatment are increasing. Metastatic bone tumor usually constitutes only part of the metastatic lesions. Although local control at the bone metastatic site alone may have little effect on the total curability, the quality of life (QOL) of the patient cannot be ensured if local control is ignored. Therefore it is important to achieve local control by interdisciplinary treatments aiming at reduced surgery and at functional conservation.

Clinical diagnosis of soft tissue tumors.

In the clinical diagnosis of a soft tissue tumor, it is important to determine whether the lesion is malignant as soon as possible. To establish such a diagnosis we perform aspiration cytology and needle biopsy simultaneously with computed radiography (CR) and echosonogram at the first visit. After the first day's investigations, it is usually possible to select the surgical procedures based on the working diagnosis in almost all patients. When the lesion is clearly benign by diagnosis, excision can be performed. For large lesions or lesions that are suspected to be malignant, CT, magnetic resonance imaging , isotope-investigation, and arteriography are also performed, effectively providing precision in an already established diagnosis. These imaging findings are useful for establishing a surgical plan by anticipating the surgical line of the safety margin. For these few patients in whom the diagnosis still remains uncertain or when preoperative treatment is planned, an incisional or excisional biopsy may be necessary to establish the final diagnosis. When preoperative treatment is performed, the effectiveness of this treatment can be evaluated by medical imaging.

Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH-cytochrome b5 reductase gene.

Hereditary methaemoglobinaemia, caused by deficiency of NADH-cytochrome b5 reductase (b5R), has been classified into two types, an erythrocyte (type I) and a generalized (type II). We analysed the b5R gene of two Thai patients and found two novel mutations. The patient with type II was homozygous for a C-to-T substitution in codon 8 3 that changes Arg (CGA) to a stop codon (TGA), resulting in a truncated b5R without the catalytic portion. The patient with type I was homozygous for a C-to-T substitution in codon 178 causing replacement of Ala (GCG) with Val (GTG). To characterize effects of this missense mutation, we investigated enzymatic properties of mutant b5R (Ala 178 Val). Although the mutant enzyme showed normal catalytic activity, less stability and different spectra were observed. These results suggest that this substitution influenced enzyme stability due to the slight change of structure. In conclusion, the nonsense mutation led to type II because of malfunction of the truncated protein. On the other hand, the missense mutation caused type I, due to degradation of the unstable mutant enzyme with normal activities in patient's erythrocytes, because of the lack of compensation by new protein synthesis during the long life-span of erythrocytes.

Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia.

Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II). We investigated the b5R gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations. The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele. From this mutant allele, the product without the catalytic portion of the enzyme is generated. The second one was a missense mutation at codon 95 (CCC-->CAC) in the other allele with the result that Pro changed to His within the flavin adenine dinucleotide (FAD)-binding domain of the enzyme. To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coll. The mutant enzyme showed less catalytic activity, less thermostability, and a greater susceptibility to trypsin than did the normal counterpart. The absorption spectrum of the mutant enzyme in the visual region differed from that of the wild-type. These results suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme. The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient.

New method of evaluating the surgical margin and safety margin for musculoskeletal sarcoma, analysed on the basis of 457 surgical cases.

The evaluation of surgical margin is useful in determining the curative success of surgical treatment of musculoskeletal sarcoma and the degree to which later surgery will be reduced by the preoperative therapy. However, until recently no reliable evaluation method has been developed for these purposes. In this paper we propose a new method for evaluating the surgical margin as drafted in 1989 by the Bone and Soft Tissue Tumor Committee of the Japanese Orthopaedic Association (JOA). In this method, surgical margin was classified into four types based on the distance between the surgical margin and the reactive zone of the tumour. These classifications of surgical margin (in order to surgical extent) are curative wide margin (curative margin), wide margin, marginal margin, and intralesional margin. The surgical margin is said to be curative if the margin is more than 5 cm outside the reactive zone. It is referred to as wide if the margin is less than 5 cm. Similarly, a margin that is in the reactive zone is considered as marginal, and a margin passing through a tumour as intralesional. Moreover, wide margin is classified as adequate (at least 2 cm outside the reactive zone) or inadequate (1 cm). In our evaluation, a "thin" barrier is considered to be a 2-cm thickness of normal tissue, a "thick" barrier as a 3-cm thickness, and joint cartilage is said to be equivalent to a 5-cm thickness. In addition, a surgical margin that is outside a barrier, with normal tissue between the barrier and the reactive zone of the tumour, is considered to be curative. This method was applied in 457 cases (involving 499 surgeries) at the Cancer Institute Hospital to determine clinical significance in the treatment of musculoskeletal sarcoma (1979-1994). From the results of this study we were able to conclude that this evaluation method can be highly useful in clinical practice for establishing optimum surgery. Moreover, we found that the safety margin for high-grade musculoskeletal sarcoma is a curative margin providing an adequate wide margin of 3 cm or more when preoperative therapy is not performed or is not effective, while the safety margin for high-grade sarcoma that responds to preoperative chemo- or radiotherapy seems to be an adequate wide margin of 2 cm. Here, radiotherapy is more effective compared to chemotherapy for reducing surgical margin. An inadequate wide margin, however, combined with radiotherapy, is not enough to ensure local curative success following surgery for musculoskeletal sarcoma.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of varicella-zoster virus genome having a PstI site in the ocular sample from a patient with acute retinal necrosis.

We detected the virus genome in ocular samples from a 65-year-old woman with clinically diagnosed acute retinal necrosis using DNA amplification. She exhibited occlusive retinal vasculitis, confluent necrotizing retinitis, mainly peripheral, and iridocyclitis. For DNA amplification, we used recently published primers specific for varicella-zoster virus (VZV) and herpes simplex virus. Using VZV primers, we detected the VZV genome in the aqueous humor, but not in the vitreous, by amplifying a DNA fragment 642 base pairs in length. HSV DNA was not detected. After detecting the VZV genome, PstI restriction endonuclease was used because an epidemiological study found that about 25% of the VZV strains in Japan carry a mutation lacking a PstI recognition site. The VZV genome from the patient had a PstI cleavage pattern, while the positive control had a VZV genome that carried a PstI-site-less mutation. We considered our patient with acute retinal necrosis to be infected with VZV having a PstI site.

Vascular mineralization in the monkey brain.

Mineralization of various degrees was found in the brains of 79 (59%) of 134 cynomolgus monkeys (Macaca fascicularis). There was no age dependency in the incidence or severity, nor were there any abnormalities in growth, weight gain, or neurologic signs, although a slight sex difference was observed. The lesions, which were basophilic and intensely positive for periodic acid-Schiff or von Kossa stain, occurred in the vascular walls of the globus pallidus in two types: globoid bodies with prominent concentric lamellar structures in and around the arteriolar and venular wall (type A) and fine granules in the media of small or medium-sized arteries (type B). Electron microscopic examination revealed dense deposits in the degenerated media of small or medium-sized arteries or the thickened walls of the arterioles. X-ray microanalysis demonstrated the presence of calcium, phosphorus, iron, zinc, magnesium, and aluminum.

Molecular basis of beta-thalassemia in Japan: heterogeneity and origins of mutations.

Characterization of beta-thalassemia mutations was attempted for 13 unrelated Japanese patients heterozygous for beta-thalassemia. We have systematically analyzed beta-thalassemia genes using polymerase-chain-reaction-related techniques; dot blot hybridization with oligonucleotide probes complementary to known mutations, restriction endonuclease assay and direct sequencing of amplified genomic DNA. Seven different mutations were detected. Six of them are an amber mutation in codon 90 (GAG to TAG), a four-base-pair deletion in codons 41 and 42 causing premature termination due to frameshift, a C-T substitution at position 654 of IVS-2, a G-A substitution at position 1 of IVS-2 and a C-G substitution at position 848 of IVS-2, leading to splicing defects, and an ocher mutation (GAA-TAA) in codon 121 causing a thalassemia intermedia phenotype with inclusion body formation in erythrocytes. A silent mutation (CTG-TTG) was also detected in codon 91 of the allele with the IVS-2 position 1 mutation. These mutations have been reported previously in the Japanese population. The other mutation is a novel one in the Japanese, an amber mutation (TGG-TAG) in codon 15, causing a beta zero-thalassemia phenotype by premature termination of the beta-globin chain synthesis. We analyzed haplotypes of chromosomes bearing each beta-thalassemia mutation. Origins and a spectrum of mutations in comparison with those detected in malaria-endemic regions are discussed.

Spontaneous globoid mineralization in the cerebellum of rats.

Cerebellar globoid mineralization in two rats was examined by light and electron microscopy, and by X-ray microanalysis. The mineralization was round to oval in shape; it varied in size and was positive for the periodic acid-Schiff and von Kossa reactions. Ultrastructurally, a concentric lamellar structure was prominent in moderately electron-dense depositions. Elemental analysis revealed the presence of large amounts of calcium and phosphorus, and small amounts of zinc, potassium and aluminum.

[Experimental studies on pasteurized autogenous bone graft].

Previous studies on hyperthermia have suggested that pasteurization makes tumors necrotic. Based on this assumption, replacement with a pasteurized autogenous bone graft after resection of a malignant bone tumor was deemed feasible for reconstruction. However, the extent of osteoinductive activity as well as mechanical strength of pasteurized bone was not clear. Consequently, experimental studies were carried out to test osteoinductivity and mechanical strength of pasteurized bone compared to autoclaved, boiled, and fresh bone. A summary of the results follows. Autoclaved ilium of rabbits resulted in degeneration of the bone material, and grafts were absorbed with little new bone formation and union. In contrast, the structures of pasteurized bones were preserved and the grafts were gradually replaced with viable bone in a manner similar to that of fresh bone graft. Bending and torsion tests of rat femurs indicated that the mechanical strength of autoclaved and boiled bones decreased significantly compared to fresh bones, whereas the mechanical strength of pasteurized bones was similar to that of fresh bones. These results demonstrated that pasteurized autogenous bone grafting can be a useful method for reconstruction after resection of malignant bone tumors.

Unusual huge intramuscular granuloma with calcium phosphate crystal deposition in the buttock.

An unusual case of a huge granuloma featuring marked calcium phosphate crystal deposition in muscle is presented. A 58-year-old male demonstrated a well-circumscribed, large, elastic firm mass which had slowly grown over 8 years in the left buttock. Clinical examinations including X-ray, ultrasonography and computed tomography all suggested a neoplastic growth. Surgical excision revealed three inter-connected irregular-shaped masses, measuring up to 11 x 10 x 6 cm in size. The histological appearance was of granulomas composed of numerous small crystals surrounded by reactive mononuclear cells embedded in fibrous tissue containing many foreign body type giant cells. Chemical analysis demonstrated the crystals to be made up of calcium phosphate.

[Surgical and preoperative treatment of soft tissue sarcoma].

Preoperative therapy effects, resected margin and local radicality were investigated in 40 cases of soft tissue sarcoma in which preoperative therapy (mainly radiation therapy) had been given. The results indicated that there was no effect upon prognosis due to postponement of surgery in order to perform preoperative treatment. In cases in which radiation therapy was used for preoperative treatment, even when a surgical margin resulted with in a tumor, no recurrence was found. However, histologically there was concern that some portion of the living tumor cell in marginal area of the tumor might have remained. Thus, at the present stage in cases having undergone initial treatment, radiation limited to the specific area of the resected margin causing non-curative margin should be given. If combined with preoperative radiation therapy, surgical intervention involving "wide margin" can be considered radical. On the other hand, cases having undergone surgery before and receiving preoperative therapy that show good response nevertheless show numerous recurrences. These recurrences, however, invariably occur outside of the irradiated area, and may be attributed to the fact that tumor cell dissemination from the earlier surgery was not within the radiation field. Hence, it was considered that the area for radiation in the case of a recurrence should extend well beyond the scar area.

Differential effects of cefmetazole sodium on the reproductive system of infant and pubertal male rats.

The effects of cefmetazole (CMZ), a cephem antibiotic which contains the N-methyltetrazolethiol (NMTT) side-chain moiety, were compared in infant (6-42 days of age) and pubertal (6-10 weeks of age) male Sprague-Dawley rats. High doses of either CMZ or free NMTT caused reductions in testicular weight and delayed maturation of spermatogenic germ cells in the testes of infant rats, implicating NMTT as the active component in causing these effects. Pubertal rats expressed neither of these effects, even when treated with doses of CMZ far in excess of those used in infant rats. The effects of CMZ and NMTT on testicular weights and histologic features of testes of rats treated as infants were mainly reversed when these animals were examined 35 and 70 days after cessation of treatment. All reproductive functional parameters were normal in mating studies using male rats which had been treated with CMZ or NMTT as infants and allowed to recover. Because of the species differences in rates of sexual maturation and the greater rate at which rats metabolize CMZ to NMTT, the relevance to humans of the testicular effects of CMZ in infant rats is unknown.

[Bolus and maintenance infusion of high-dose methotrexate].

For 17 patients with bone and soft tissue sarcoma, 32 bolus and maintenance infusions of high-dose methotrexate (MTX) were performed to induce rapid elevation of plasma MTX concentration and to maintain it. At the doses of 200-300 mg/kg, plasma MTX concentrations were measured by enzyme assay. It needed four hours to reach the steady state by five hours' constant rate infusion, with the plasma plateau concentration at about 1 X 10(-3) M/L. On the other hand, bolus infusion of MTX needed only one hour to reach over 5 X 10(-4) M/L, and the plateau concentration lasted during the maintenance infusion period. By the constant rate infusion as well as bolus infusion, it was possible to maintain the plasma concentration over 1 X 10(-4) M/L for 12 hours, but both infusions can not maintain 1 X 10(-3) M/L for a long time. From these results, clinical effects of high dose MTX against sarcomas seemed to be evaluated by the plasma MTX plateau concentration and the maintenance time.

[Hyoid bone position and airway accompanied with influence of head posture].

The position of hyoid bone has been analyzed using roentgenogram. But, due to the great variability with the change of head posture, the position could not be determined correctly. In the present study, we investigated the influence of head posture to the position of hyoid bone. Subjects consisted of 32 Japanese males, those mean age was 25.8 years. Lateral cephalograms had been taken for each subjects at 5 different head posture. The findings were as follows. 1. Superimposed on SN plane, hyoid bone moved along the line of menton to 4th cervical vertebrae (C4) with the change of head posture. The hyoid bone moved in parallel with the line of 3rd cervical vertebrae (C3) to C4 (cervical plane). In spite of these movements, RH kept an uniform ratio. 2. The distances between sella turcica and hyoid bone and between C3 and hyoid bone, and the angle between menton, porion and hyoid bone, were at constant value. 3. Some measurements included the distance between hyoid bone and menton and the angle between nasion, sella turcica, hyoid bone showed very high correlation with the change of head posture. 4. EpPW1 (the width of airway) and the distance from C4 to hyoid bone showed almost the same change. It means the position of hyoid bone has a great influence on airway.

[Preoperative radiotherapy of bone and soft tissue sarcomas].

UNLABELLED: The role of preoperative radiotherapy was evaluated in 16 cases with soft tissue sarcoma and 13 cases with osteosarcoma. Nine osteosarcoma cases underwent radiotherapy of whole lesion, and 4 cases had radiotherapy only of the surgically incurable portion. There were no local recurrences in M0 cases, but skin necrosis occurred in the whole radiation group. As for the soft tissue sarcomas, local recurrence was not seen in virgin cases, but two cases which had received previous treatment showed local recurrence. There were no cases with severe side effects. CONCLUSION: 1. Partial radiotherapy was effective as preoperative treatment for osteosarcoma. 2. preoperative radiotherapy is better than postoperative radiotherapy from many standpoints.

[Evaluation method of surgical margin in musculoskeletal sarcoma].

The evaluation of surgical margin is useful in determining the curability of surgical treatment of musculoskeletal sarcoma and the degree of possibility to reduce later surgery as part of preoperative therapy. However, at present there has been no worthwhile evaluation method developed for these purposes. In this paper we propose a new evaluation system for surgical margin as drafted by the Bone and Soft Tissue Tumor Committee of JOA. This system was applied in 197 cases at the Cancer Institute Hospital to determine the clinical significance in the treatment of musculoskeletal sarcoma. In this method, surgical margin was classified into four types according to the distance between the surgical margin and the reactive zone of the tumor. The types of surgical margins (in order of surgical extent) are curative margin, wide margin, marginal margin, and intralesional margin. Surgical margin is said to be curative if the margin is more than 5cm outside the reactive zone. It is referred to as wide if the margin is less than 5cm and marginal if the margin is at the reactive zone. In our evaluation, a thin barrier is estimated as a 2cm thickness of normal tissue, a thick barrier as a 3cm thickness of normal tissue, and joint cartilage as 5cm thickness. Moreover, in all cases the surgical margin is considered to be curative if the margin is outside a barrier and there is normal tissue between the barrier and the reactive zone of the tumor. The results of the study were as follows.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical safety studies of cefmetazole.

In order to assess the safety of cefmetazole, preclinical multiple-dose parenteral studies, varying from one to three months in length, were conducted in Sprague-Dawley rats and beagle dogs. Although the largest doses used were in multiples of several times the weight-adjusted doses intended for humans, cefmetazole was generally well tolerated. The principal adverse effect noted in the adult rats receiving the largest doses (2000 and 2500 mg/kg/day) of cefmetazole was slight elevation of serum alanine aminotransferase. Infant rats injected subcutaneously with 300 mg/kg/day or more of cefmetazole for 35 consecutive days had reversible reductions in testicular weight and maturation of spermatogenesis, but not lasting discernible effect on reproductive function. The most consistent effects of longterm multiple dosing with cefmetazole in dogs consisted of vomiting and retching during dosing and reversible haematological changes (mild regenerative anaemia, positive Coombs' test, clinically-silent thrombocytopenia) in a number of the dogs. These findings supported the interpretation that cefmetazole was acceptably safe for clinical studies in humans.