Changes in the basement membrane zone components during skeletal muscle fiber degeneration and regeneration.

The basement membrane of skeletal muscle fibers is believed to persist unchanged during myofiber degeneration and act as a tubular structure within which the regeneration of new myofibers occurs. In the present study we describe macromolecular changes in the basement membrane zone during muscle degeneration and regeneration, as monitored by immunofluorescence using specific antibodies against types IV and V collagen, laminin, and heparan sulfate proteoglycan and by the binding of concanavalin A (Con A). Skeletal muscle regeneration was induced by autotransplantation of the extensor digitorum longus muscle in rats. After this procedure, the myofibers degenerate; this is followed by myosatellite cell activation, proliferation, and fusion, resulting in the formation of new myotubes that mature into myofibers. In normal muscle, the distribution of types IV and V collagen, laminin, heparan sulfate proteoglycan, and Con A binding was seen in the pericellular basement membrane region. In autotransplanted muscle, the various components of the basement membrane zone disappeared, leaving behind some unidentifiable component that still bound Con A. Around the regenerated myotubes a new basement membrane (zone) reappeared, which persisted during maturation of the regenerating muscle. The distribution of various basement membrane components in the regenerated myofibers was similar to that seen in the normal muscle. Based on our present and previous study (Gulati, A.K., A.H. Reddi, and A.A. Zalewski, 1982, Anat. Rec. 204:175-183), it appears that some of the original basement membrane zone components disappear during myofiber degeneration and initial regeneration. As a new basement membrane develops, its components reappear and persist in the mature myofibers. We conclude that skeletal muscle fiber basement membrane (zone) is not a static structure as previously thought, but rather that its components change quite rapidly during myofiber degeneration and regeneration.

Cryptococcal meningitis with an antecedent cutaneous Cryptococcal lesion.

Cutaneous cryptococcosis, caused by an encapsulated yeast, Cryptococcus neoformans, is generally associated with concomitant systemic infection. Here we report a case of primary cutaneous cryptococcosis with spread to central nervous system in an HIV seronegative young boy. In the present case, a 17-year-old boy who was suffering from a non-healing ulcer on his right great toe for 5 months, presented with the signs and symptoms of meningitis. Cryptococcus neoformans var. gattii was isolated from the CSF of the patient. Amphotericin B administration produced recovery from the meningitis as well as from the ulcer. This case study suggests that primary cutaneous cryptococcosis can be diagnosed provisionally by a simple Gram stained smear and India ink examination in order to avoid occurrence of disseminated cryptococcosis, including meningial involvement, which may have a fatal outcome.

Prevalence of Helicobacter pylori in asymptomatic subjects--a nested PCR based study.

The aim of the study was to see the prevalence of Helicobacter pylori in asymptomatic children and adults by using nested PCR which is considered to be more specific than serological methods. Saliva and stool samples of 137 healthy children (aged 8 months to 16 y) and 108 asymptomatic adults (aged 17-60 y) were collected. PCR with primers targeting Hsp60 gene sequence of H. pylori was used. H. pylori positivity with nested PCR was observed in 45.7% (112/245) of the saliva and 42.8% (105/245) of the stool specimens. Prevalence of H. pylori in saliva was found to be 2.1%, 22.7%, 55.9%, 56.0%, 68.9% and 62.9% in the age groups of < 5 y, 6-10 y, 11-16 y, 17-30 y , 31-45 y and 45-60 y, respectively. The detection rates in stool were 4.25% in < 5 y, 13.64% in 6-10 y, 50% in 11-16 y, 64% in 17-30 y, 58.62% in 31-45 y and 61.1% in 45-60 y of age groups. The most favourable age group for acquiring the infection was 11-16 y. H. pylori positivity increased with lowering of socioeconomic status. There was no gender bias in prevalence of the bacterium.

Hematological profile of HIV patients in relation to immune status - a hospital-based cohort from Varanasi, North India.

OBJECTIVES: To study the spectrum of hematological manifestations and evaluate the relationship between various hematological manifestations and CD4 cell counts in a hospital-based cohort of HIV-infected adults in and around Varanasi, North India. MATERIALS-METHODS: The clinical and hematological profiles of the patients attending the Infectious Disease Clinic, Varanasi, India were recorded. The relationship between CD4 counts and various hematological manifestations was analyzed. RESULTS: A total of 470 HIV-infected individuals were followed for 830 person years of observation (PYO). Rate of hematological episodes was 1047 episodes per 1000 PYO. CD4 counts were significantly lower in individuals with severe anemia and neutropenia compared to those without. However, no relation could be established between thrombocytopenia and CD4 counts. In the above- mentioned population, CD4 levels were significantly lower in those with anemia/neutropenia harboring any particular disease compared to those who had the same disease without anemia/ neutropenia. CONCLUSIONS: There is a strong negative association between CD4 counts and the severity of anemia and neutropenia in this population. They can be considered as good clinical indicators to predict and access the underlying immune status. Though fall in the CD4 levels during neutropenia is observed, it is difficult to comment since the estimations of CD4 rely on the total leukocyte counts. However, the relation between anemia and disease progression is straight forward and quite useful for the treating physician.

Pulmonary fungal ball in non-immunocompromised patient: a case report.

Fungal ball caused by Aspergillus species is an opportunistic infection. We describe a case report of a patient with culture positive Aspergillus fumigatus who presented with complaints of cough and expectoration with recurrent episodes of haemoptysis. Tuberculosis is the commonest cause of haemoptysis in India. However fungal ball is also one of the leading cause of haemoptysis. Hence laboratory evaluation of haemoptysis should not only include work up for tuberculosis but sample should also be submitted for mycological evaluation.

Acanthamoebae presenting as primary meningoencephalitis in AIDS.

A rare case of Acanthamoebae meningoencephalitis is diagnosed in cerebrospinal fluid (CSF) of a 24 years old male suffering from acquired immunodeficiency syndrome (AIDS) patient on the basis of bright field microscopy and culture growth on non-nutrient agar with Escherichia coli. This case illustrates that Acanthamoebae should be considered in the differential diagnosis of meningoencephalitis in AIDS in addition to tuberculosis and cryptococcus infection in tropical areas.

Diarrhea, CD4 counts and enteric infections in a hospital - based cohort of HIV-infected patients around Varanasi, India.

BACKGROUND: As most of the studies in HIV patients with diarrhea were cross sectional, focusing on the etiological agents, we are reporting data on the rate of diarrhea, associations between diarrhea and CD4 counts and variation in frequency of identifying a pathogen with consistency of diarrhea and duration in a prospective hospital based study. METHODS: Stool specimens were obtained between Jan 2001 and April 2003 from HIV infected adults with diarrhea presenting to Infectious Disease clinic, Banaras Hindu University, Varanasi. In all patients with diarrhea, specimens were examined by microscopy and cultures to identify pathogens. RESULTS: During the study, 630 person years of observations with diarrhea were analyzed. 140 stool samples were collected representing 43% of episodes of reported diarrhea. Positivity of finding a pathogen from watery stools and formed stools were 40%&24% respectively (p < 0.01) probably due to associated inflammation is more in watery diarrhea. Patients having chronic diarrhea are 2.25 (95%CI 1.52-2.81) times at more risk of developing other opportunistic infections compared to those who don't have. However this is not true with the acute diarrhea where risk of harboring the opportunistic infections remain same. CONCLUSION: Diarrhea was most strongly associated with low CD4 counts. Over two-thirds of diarrheal episodes were undiagnosed, suggesting that unidentified agents or primary HIV enteropathy are important causes of diarrhea in this population. There is a strong negative association between duration of diarrhea and CD4 levels.

Failure of host axons to regenerate through a once successful but later rejected long nerve allograft.

Host axons will regenerate through a long nerve allograft in an immunologically tolerant rat. However, if tolerance is abolished, rejection occurs and allogeneic cells (e.g., Schwann, vascular, perineurial, etc.) as well as regenerated host axons disappear from the allograft. Because following tolerance abolition host axons begin to regenerate into the connective tissue remnants of the rejected nerve allografts, the extent of this renewed axonal growth was investigated. It was found that in a tolerance-abolished rat, host axons only regrew in to the proximal 1 cm of a 4-cm allograft which in a fully tolerant recipient would have had numerous allogeneic Schwann cell-myelinated axons throughout its length. It is concluded that viable allogenic cells (i.e., Schwann, fibroblast, and vascular) together with their connective tissue matrix provide the best way to aid host nerve fiber regeneration through a long nerve allograft.

Immunological fate of Schwann cell-populated acellular basal lamina nerve allografts.

Acellular basal lamina allografts are known to exhibit reduced immunogenicity, but the extent of host axonal regeneration through such grafts is reduced in comparison to the cellular isografts. The present study was designed to determine both the ability of cultured Schwann cells to populate acellular allografts, and the effects of Schwann cells on immunogenicity and regenerative potential. Isogeneic and allogeneic Schwann cell-populated acellular allografts of 2.0 cm length were used to repair a surgically created gap in the host rat peroneal nerve. Transplanted nerves were analyzed at 1, 2, 4, and 8 weeks later to determine their fate and ability to support axonal regeneration. Acellular allografts cocultured with isogeneic Schwann cells survived and supported axonal regeneration through them. In contrast, acellular allografts cocultured with allogeneic Schwann cells underwent rejection and were unsuccessful. The results show that cultured Schwann cells continue to exhibit immunogenicity and are a critical factor in allograft survival. It is thus important to use host-matched (isogeneic or autologous) Schwann cells to populate acellular basal lamina grafts in order to enhance their axonal growth-supporting function.

Histochemical localization of gamma glutamyl transpeptidase in the paranodal region of peripheral nerve.

Gamma glutamyl transpeptidase (GGT), an amino acid transport enzyme, was investigated in normal and degenerated sciatic nerve of rat. The enzyme activity, which is considered to be a marker for cerebrovascular endothelium, was found to be absent in microvessels of normal and degenerated nerves. In the perineurium of normal nerve, GGT activity was faint, while in degenerated nerve, it increased. The most striking finding of this study was the observation of GGT activity at the paranode of each normal myelinated axon. It is interesting that after axotomy (8 weeks), no GGT activity was observed in the Schwann cells of degenerated nerve. Thus, Schwann cell plasmalemma contributed to GGT staining only when this cell was in contact with an axon mature enough to cause it to produce myelin. We conclude that, in peripheral nerve, transmembrane amino acid transport is apparently regional and associated with the paranodal region of myelinated nerve fibers.

A comparison of lectin binding in rat and human peripheral nerve.

Eleven different fluorescein- or peroxidase-conjugated lectins with different sugar-binding affinities were employed to analyze and compare glycoconjugates of rat and human peripheral nerves at the light microscopic level. A majority of lectins showed a distinct binding pattern in different structures of the nerve. Lectin binding was similar but not identical in rat and human nerves. Limulus polyhemus agglutinin did not stain any structures in rat or human nerves. In both species, all other lectins bound to the perineurium. Perineurial staining was intense with Canavalia ensiformis (Con A), Triticum vulgaris (WGA), Maclura pomifera (MPA); moderate with Glycine max (SBA), Griffonia simplicifolia-I (GS-I) and GS-II; weak with Ulex europaeus (UEA), Dolichos biflorus (DBA), and Ricinus communis (RCA). In the endoneurium of both species, ConA staining was intense, MPA and WGA moderate, SBA, GS-II, PNA, and RCA weak, and UEA and DBA absent. Interestingly, GS-I stained rat but not human endoneurium. Most lectins bound to blood vessels. GS-I bound to rat but not human, whereas UEA bound to human but not rat vessels. The results show that lectins can be used to reveal heterogeneity in sugar residues of glycoconjugates within neural and vascular components of nerves. They may therefore be potentially useful in detecting changes in glycoconjugates during nerve degeneration and subsequent regeneration after trauma or in pathological states.

Expression of phosphatidylinositol 3-kinase during EGF-stimulated wound repair in rabbit corneal epithelium.

PURPOSE: To investigate the effect of epidermal growth factor (EGF) on the induction of phosphatidylinositol 3-kinase (PI 3- kinase) gene expression during rabbit corneal epithelial wound repair. METHODS: Epithelial wounds (6 mm in size) were created in rabbit corneas and EGF (2 microg) applied every 8 hours to one eye, and the other eye served as a control. The wound repair was monitored by staining the tissue with fluorescein followed by photography. The wound area was quantified with a computer program. At different time intervals, the rabbits were killed and the corneal epithelium used for estimation of PI 3-kinase activity, western blot analysis, or reverse transcription-polymerase chain reaction (RT-PCR). For in situ hybridization, the whole corneas were sectioned and the sections processed with PI 3-kinase mRNA probes. RESULTS: In the untreated eye, the epithelial wound progressively healed in a time-dependent manner, with 75% of the wound closed at 48 hours post wounding. Application of EGF to the corneal epithelium further stimulated wound repair at all time intervals, and the wound was completely closed at 48 hours. Analysis of PI 3-kinase showed a time-dependent increase in its enzyme activity that was maximally increased at 36 hours, the time when the wound was nearly closed. Western blot analysis revealed increased amounts of PI 3- kinase protein during the course of wound repair. Analysis of RT-PCR products from epithelial tissues, taken at different times during wound repair, showed increased PI 3-kinase expression that was maximum at 48 hours post wounding. A visible increase in PI 3-kinase gene expression was also detected by in situ hybridization during the course of the wound repair. This expression was increased maximally by EGF at 48 hours post wounding. CONCLUSIONS: The results indicate a temporal correlation between increased activation and expression of PI 3- kinase and the epithelial wound repair. Topical application of EGF further stimulates the activity and expression of PI 3- kinase. It is suggested that PI 3- kinase and its products may play a role in EGF-induced cell proliferation during corneal epithelial wound repair.

Restoration of denervated skeletal muscle transplants after reinnervation in rats.

The present study describes reinnervation and restoration of rat skeletal muscle denervated for the duration of 3, 6 or 12 months. Denervation of extensor digitorum longus (EDL) muscle was achieved by cutting and ligating the donor rat sciatic nerve in situ. At 3, 6 and 12 months, the denervated EDL muscles were removed and transplanted into an innervated normal leg of another rat. In addition, normal (i.e., no prior denervation) muscles were transplanted as controls for comparison. The muscles were analyzed at 4 and 12 weeks after transplantation. The EDL muscle weight and myofiber size decreased with extended denervation times. After transplantation, the muscles underwent regeneration and reinnervation, and recovered as determined by an increase in muscle mass and myofiber size. The 3-month denervated muscle regenerates recovered completely, and were similar to the non-denervated normal muscle regenerates. Reinnervation, and partial recovery of muscle weight and myofiber size was observed in 6- and 12-month denervated muscle transplants. These results document that while regeneration and reinnervation does occur in denervated muscles after transplantation, the extent of recovery is related to the duration of denervation.

Chronic spinal cord transection does not affect peripheral nerve regeneration.

Spinal cord transection is known to cause progressive changes in motor neurons and hind limb muscles. In the present study, regeneration of the peroneal nerve was examined in rats 25 weeks after a T9 spinal cord transection. Successful regeneration and innervation of the target muscle was observed after crush injury to the nerve in the spinal cord transected animals. It is concluded that the ability of peripheral nerve to regenerate remains preserved after spinal cord injury.

Peripheral nerve regeneration through short- and long-term degenerated nerve transplants.

The present study was designed to compare regenerative potential of normal and degenerated nerve grafts. Peripheral nerves in rats were induced to undergo in situ degeneration for a period of 6 weeks, 3, 6 and 12 months. During early phase of denervation the myelin and axons degenerated and were absorbed. With prolonged denervation (i.e. 12 months), such nerves were reduced in size and exhibited extensive fibrosis. A 2 cm long segment of the degenerated nerve was transplanted in an surgically created gap in the host peroneal nerve to evaluate their regeneration supporting ability. Regeneration of host axons occurred rapidly through nerves degenerated for a period up to 3 months. The extent of regeneration was compromised in 6-month degenerated nerve group, and was significantly reduced in the 12-month degenerated nerve grafts. These results show that with extended degeneration interval, the regeneration supporting ability of nerves is compromised. It is concluded that nerve repair should not be excessively delayed in order to compromise recovery.

The influence of cultured Schwann cells on regeneration through acellular basal lamina grafts.

Acellular basal lamina grafts have been shown to be less immunogenic in comparison to cellular grafts, but possess a limited potential for supporting axonal regeneration through them. The present study describes the effect of cultured Schwann cells on enhancing regeneration through acellular grafts. 2 cm long acellular grafts, and in vitro Schwann cell populated acellular grafts were used to repair a surgically created gap in the host peroneal nerve. The transplants were analyzed at 1, 2, 4 and 8 weeks to determine their ability to support axonal regeneration. Host axonal regeneration through Schwann cell cocultured acellular grafts occurred rapidly and was significantly better as compared to non-cultured acellular grafts. The results demonstrate a beneficial effect of Schwann cell culture pretreatment on regeneration through acellular grafts and an improved recovery of the target muscle. The procedure of first preparing acellular grafts with subsequent coculture with Schwann cells offers a novel approach for the repair of injured nervous tissue.

The effect of X-irradiation on skeletal muscle regeneration in the adult rat.

Muscle regeneration was induced by transplanting the extensor digitorum longus (EDL) muscle in adult rats to examine the effect of X-irradiation on muscle regeneration. The EDL muscles were removed, irradiated with X-rays to administer 650 R, 2,000 R or 10,000 R, and transplanted into the original animal and location. Muscles from non-irradiated control group and each irradiated group were analyzed morphologically at 4, 7, 14 and 30 days post-transplantation. The regeneration pattern in the non-irradiated and 650-R irradiated muscles was similar. A majority of myofibers underwent degeneration followed by regeneration from the precursor myosatellite cells. The myosatellite cells proliferated, differentiated into myoblasts and then fused to form myotubes and myofibers. Muscles exposed to 2,000 R underwent initial degeneration and myosatellite cell activation, however, considerably fewer myotubes regenerated in these muscles. In muscles exposed to 10,000 R, again myofiber degeneration and myosatellite cell activation was evident, but these cells remained undifferentiated and did not fuse to form myotubes. These results show a dose-dependent inhibition in muscle regeneration due to irradiation.

Evaluation of acellular and cellular nerve grafts in repair of rat peripheral nerve.

Nerve grafts composed of basal lamina scaffolds and lacking viable Schwann cells have recently been shown to be effective in supporting axonal regeneration. As only short grafts were used in those studies, the present investigation was conducted to evaluate the ability of long acellular basal lamina nerve grafts and equivalent cellular grafts to support axonal regeneration for nerve gap repair. Cellular grafts consisted of nerve segments that had degenerated in situ for 4 weeks. Acellular grafting material consisted of similar segments that were repeatedly frozen and thawed to kill all cells prior to grafting. The results show that host axons can regenerate through the entire 4-cm length of cellular grafts but not through acellular basal lamina grafts. However, in the acellular grafts numerous axons were seen in the proximal 2-cm region. It is concluded that basal lamina grafts possess limited ability to support axonal regeneration. As in cellular grafts, viable Schwann cells appear to be important for regeneration to occur over longer distances.

Axon regeneration through blood vessel allografts after cyclosporine treatment.

The present study describes the usefulness of blood vessel allografts to repair gaps in rat peripheral nerve after immunosuppression with cyclosporine. Isogeneic strains of rats with known histoincompatibility were used for this study. A 10-mm gap was created in the peroneal nerve of host Fischer rats. The gap was bridged by a 12-mm section of internal carotid artery removed from a Buffalo strain of rat. The host rats were divided into two groups. One group received no immunosuppression, whereas the other group was treated with cyclosporine. Untreated control rats immunologically rejected the allografted vessels and were unable to support host axonal regeneration through them. On the other hand, in cyclosporine-treated rats the allografted vessels survived. The regenerating host axons reorganized to form a functional nerve within the vessel conduit. The regenerated axons persisted even after rejection of the allografted vessel caused by cessation of immunotherapy. These results show that blood vessel allografts can serve as an effective conduit for reorganization of regenerated nerves and can bridge gaps in peripheral nerves.

Survival of nerve allografts in sensitized rats treated with cyclosporin A.

This study examined the effect of immunosuppressive treatment with cyclosporin A (Cy-A) on the survival of nerve allografts in sensitized rats. Nerve- or skin-sensitized untreated rats rejected a second nerve allograft of the same genotype as the first in an accelerated manner. In this situation, only a few host axons grew into the proximal 1 cm of a 4 cm-long nerve allograft. However, if sensitized rats were given Cy-A (10 mg/kg daily), the second nerve allograft survived, and numerous host axons regenerated through the 4-cm length of the allograft. These results indicated that Cy-A was an effective immunosuppressive agent in sensitized rats. We conclude that, in rats, donor-specific sensitization is not a contraindication to the use of nerve allografts to aid in the repair of injured nerve when Cy-A is used for immunosuppression.